U.S. patent application number 10/514781 was filed with the patent office on 2006-12-14 for toxin-related antibodies with antimicrobial and antiviral activity.
Invention is credited to Luisa Bracci, Antonio Cassone, Luisa Lozzi, Neri Paolo, Luciano Polonelli.
Application Number | 20060280750 10/514781 |
Document ID | / |
Family ID | 9936462 |
Filed Date | 2006-12-14 |
United States Patent
Application |
20060280750 |
Kind Code |
A1 |
Polonelli; Luciano ; et
al. |
December 14, 2006 |
Toxin-related antibodies with antimicrobial and antiviral
activity
Abstract
Anti-idiotypic antibodies which recognise the idiotope of an
antibody specific for a yeast killer toxin possess microbicidal
activity. Fragments (e.g. decapeptides) of these anti-idiotypic
antibodies, particularly those comprising CDR residues, also show
microbicidal activity, as do peptides having 5 the same sequence
but composed of D-amino acids, or including amino acid
substitutions. Peptidomimetics of these microbicidal polypeptides
are also provided. Antiviral activity is also seen.
Inventors: |
Polonelli; Luciano; (Parma,
IT) ; Cassone; Antonio; (Roma, IT) ; Bracci;
Luisa; (Siena, IT) ; Paolo; Neri; (Siena,
IT) ; Lozzi; Luisa; (Siena, IT) |
Correspondence
Address: |
Albert Wai-Kit Chan;Law Offices of Albert Wai-Kit Chan
World Plaza Suite 604
141-07 20th Avenue
Whitestone
NY
11357
US
|
Family ID: |
9936462 |
Appl. No.: |
10/514781 |
Filed: |
May 9, 2003 |
PCT Filed: |
May 9, 2003 |
PCT NO: |
PCT/IB03/02348 |
371 Date: |
March 2, 2005 |
Current U.S.
Class: |
424/159.1 ;
424/164.1; 424/178.1; 435/320.1; 435/326; 435/69.1; 514/44R;
530/324; 530/328; 530/388.3; 530/388.4; 530/391.1; 536/23.53 |
Current CPC
Class: |
A61P 31/12 20180101;
Y02A 50/41 20180101; C07K 19/00 20130101; A61K 2039/505 20130101;
Y02A 50/484 20180101; A61P 31/04 20180101; Y02A 50/30 20180101;
A61P 31/10 20180101; C07K 14/39 20130101; C07K 2319/00 20130101;
C07K 2317/622 20130101; C07K 16/4208 20130101 |
Class at
Publication: |
424/159.1 ;
424/164.1; 424/178.1; 435/069.1; 435/320.1; 435/326; 530/388.3;
530/388.4; 530/391.1; 514/044; 536/023.53; 530/324; 530/328 |
International
Class: |
C07K 16/12 20060101
C07K016/12; C07K 16/10 20060101 C07K016/10; C07K 16/08 20060101
C07K016/08; C07K 16/46 20060101 C07K016/46; C07K 7/08 20060101
C07K007/08; A61K 39/42 20060101 A61K039/42; A61K 39/40 20060101
A61K039/40; C12P 21/06 20060101 C12P021/06; C07H 21/04 20060101
C07H021/04; C12N 5/06 20060101 C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2002 |
GB |
0210783.7 |
Claims
1. A polypeptide comprising: at least one amino acid sequence which
is a fragment of at least x amino acids from the amino acid
sequence of a variable region of an anti-idiotypic antibody which
recognizes the idiotope of an antibody specific for a yeast killer
toxin, optionally with y amino acids within said x amino acids
being substituted by different amino acid(s), wherein x is at least
3 and y is at least 1.
2. The polypeptide of claim 1, consisting of at least 3 amino
acids.
3. The polypeptide of claim 1 consisting of at most 90 amino
acids.
4. The polypeptide of claims consisting of between 8 and 20 amino
acids.
5. The polypeptide of claim 4, consisting of 10 amino acids.
6. The polypeptide of claim wherein x is at least 8.
7. The polypeptide of claim 6, wherein x is 10.
8. The polypeptide of claim wherein at least 1 amino acid within
said x amino acids is substituted by different amino acid(s).
9. The polypeptide of claim 1, wherein the fragment of at least x
amino acids preferably includes at least 1 amino acid from a CDR
within the antibody.
10. The polypeptide of claim 1, comprising L amino acids and/or D
amino acids.
11. The polypeptide of claim comprising sequence
AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.sub.7-AA.sub.8-A-
A.sub.9-AA.sub.10, wherein: each of AA.sub.1 . . . AA.sub.10 is
independently a D- or L- amino acid; AA.sub.1 is E, A or G;
AA.sub.2 is K, A or G; AA.sub.3 is V, A or G; AA.sub.4 is T, A or
G; AA.sub.5 is M, A or G; AA.sub.6 is T, A or G; AA.sub.7 is C, S,
A or G; AA.sub.8 is S, A or G; AA.sub.9 is A or G; and AA.sub.10 is
S, A or G; provided that no more than 4 of AA.sub.1, AA.sub.2,
AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.6, AA.sub.7, AA.sub.8,
AA.sub.9, and AA.sub.10 are A; and provided that no more than 2 of
AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.6,
AA.sub.7, AA.sub.8, AA.sub.9, and AA.sub.10 are G.
12. The polypeptide of claim 1, comprising amino acid sequence SEQ
IDs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 20,
23, 24, 25, 26, 27, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,
44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70 or 71, with constituent amino
acids in the D- and/or L- configuration.
13. The polypeptide of claim 12, consisting of SEQ ID 3, 4, 23, 27
or 33.
14. A polypeptide consisting of SEQ ID 4 or SEQ ID 33, and
consisting of D-amino acids.
15. A microbicidal peptidomimetic compound which is isosteric with
respect to the polypeptide of claim 1.
16. An anti-idiotypic antibody which recognizes the idiotope of an
antibody specific for a yeast killer toxin, with the proviso that
the anti idiotypic antibody is not the K10 rat monoclonal
antibody.
17. An antibody which recognizes the idiotope of an antibody of
claim 16.
18. The antibody of claim 16 wherein the antibody is a single-chain
Fv antibody.
19. The antibody of claim 18, wherein the antibody is H6 or
H20.
20. The antibody of claim 16, wherein the antibody is a monoclonal
antibody.
21. The antibody of claim 20, wherein the antibody is a human or
humanized antibody.
22. The antibody of claim 21, wherein the antibody comprises one or
more CDR from H6, H20 or K20.
23. The antibody of claim 16 wherein the toxin is the killer toxin
from P. anomala.
24. A polypeptide comprising: at least one amino acid sequence
which is a fragment of at least x amino acids from the amino acid
sequence of a variable region of an antibody of claim 16,
optionally with y amino acids within said x amino acids being
substituted by different amino acid(s), wherein x is at least 3 and
y is at least 1.
25. Nucleic acid that encodes a polypeptide of claim 1, and/or an
antibody which recognizes the idiotope of an antibody specific for
a yeast killer toxin, with the proviso that the anti idiotypic
antibody is not the K10 rat monoclonal antibody.
26. The antibody of claim 16 or the polypeptide comprising: at
least one amino acid sequence which is a fragment of at least x
amino acids from the amino acid sequence of a variable region of an
anti-idiotypic antibody which--recognizes the idiotope of an
antibody specific for a yeast killer toxin, optionally with y amino
acids within said x amino acids being substituted by different
amino acid(s), wherein x is at least 3 and y is at least 1 which
has antimycotic activity and/or antibiotic activity.
27-34. (canceled)
35. A pharmaceutical composition comprising (a) the antibody of
claim 16 and (b) a pharmaceutical carrier.
36. A pharmaceutical composition comprising (a) the polypeptide of
claim 1 and (b) a pharmaceutical carrier.
37. A pharmaceutical composition comprising (a) the peptidomimetic
of claim 15 and (b) a pharmaceutical carrier.
38. A pharmaceutical composition comprising (a) the nucleic acid of
claim 25 and (b) a pharmaceutical carrier.
39. A method for treating a patient suffering from a microbial
and/or viral infection, comprising administering to the patient the
pharmaceutical composition of claim 35.
40. A method for treating a patient suffering from a microbial
and/or viral infection, comprising administering to the patient the
pharmaceutical composition of claim 36.
41. A method for treating a patient suffering from a microbial
and/or viral infection, comprising administering to the patient the
pharmaceutical composition of claim 37.
42. A method for treating a patient suffering from a microbial
and/or viral infection, comprising administering to the patient the
pharmaceutical composition of claim 38.
Description
TECHNICAL FIELD
[0001] This invention is in the field of microbicides and
antivirals, in particular those derived from yeast killer
toxins.
BACKGROUND ART
[0002] Killer toxins (KTs) are proteins secreted by yeasts which
are able to kill other yeasts or microorganisms which compete in
nature for the same ecological niche [1]. Although they are
attractive therapeutic tools, due to their wide spectrum of
microbicidal activity, they are of no practical use because of
their instability in the host physiological milieu as well as their
antigenicity and toxicity. Instead, the use of anti-idiotypic
antibodies which mimic KTs has been shown to be effective.
[0003] The killer toxin from Pichia anomala (`PaKT` ) has a wide
spectrum of microbicidal activity against pathogens including
Candida albicans, Aspergillus fumigatus, Pneumocystis carinii,
Mycobacterium tuberculosis, Pseudomonas aeruginosa, and
Staphylococcus aureus [2, 3, 4]. This observation has been
exploited by the generation of a PaKT-neutralizing monoclonal
antibody in mice (mAb KT4) [5] whose idiotype (Id) is able to
induce the production of anti-idiotypic antibodies (antilds) [6, 7,
8]. These antilds represent the internal image of the active PaKT
domain and as such exert its biological activities, including
binding to the PaKT receptor (KTR) of susceptible microorganisms
and broad spectrum microbicidal activity overlapping that of PaKT
(FIG. 1).
[0004] Experimental animals in which these antibodies (`KTIdAb )
are raised by idiotypic vaccination with mAb KT4 have repeatedly
been shown to be protected against mucosal or systemic challenges
by C.albicans [7, 8]. There is also ample evidence of
susceptibility in vitro to KTIdAb by diverse microbial pathogens
such as M.tuberculosis (including multidrug resistant strains),
P.carinii, and others [4, 9].
[0005] Idiotypic theory (FIG. 1) also predicted that antibodies
against PaKT receptors would mimic PaKT activity. This has been
demonstrated in animals and humans during the course of
experimental and natural infections caused by PaKT-sensitive
microorganisms [10]. Human natural anti-KTR antibodies have been
shown to have microbicidal activity in vitro against C.albicans,
M.tuberuclosis, and P.carinii, to inhibit P.carinii infectivity of
nude rats, and to be protective against passive transfer in vivo,
in an experimental model of rat vaginal candidiasis [4, 10,
11].
[0006] Based on these results, and in order to obtain standard
KTldAb in sufficient amounts, rat monoclonal IgM (mAb K10) and
mouse single-chain Fv (scFv H6) microbicidal antibodies have been
obtained [12, 13]. These two antibodies have strong microbicidal
effects in vitro against important pathogenic microorganisms
including: C.albicans [12, 13]; C.krusei and C.glabrata (including
fluconazole-resistant strains); Cryptococcus neoformans; A.
fumigalus [14]; M.tuberculosis [4]; S.aureus, Enterococcus
faecalis, E.faecium, and Streptococcus pneumoniae (including
methicillin-, vancomycin- and penicillin-resistant strains) [15],
S.mutans, Leishmania major, L.infantum and Achantamoeba castellani.
Furthermore, they showed specific therapeutic activity in an
experimental model of rat vaginal candidiasis by intravaginal
administration [13]. In addition, K10 proved to be therapeutic
against P.carinii pneumonia in rats infected by aerosol
administration [16], and in mice transplanted with T cell depleted
bone marrow against aspergillosis caused by nasal instillation
[14].
[0007] Although the existence of scFv H6 has been reported, a
method for its manufacture has not previously been disclosed, and
nor has its amino acid sequence.
[0008] It is an object of the invention to provide further and
improved antimicrobial and/or antiviral compounds.
DISCLOSURE OF THE INVENTION
[0009] Antibodies of the Invention
[0010] The invention provides an anti-idiotypic antibody which
recognises the idiotope of an antibody specific for a yeast killer
toxin, with the proviso that the anti-idiotypic antibody is not the
K10 rat monoclonal antibody. The antibody preferably has
microbicidal activity (e.g. it retains yeast killer toxin activity)
and/or antiviral activity.
[0011] An anti-idiotypic antibody of the invention can be used to
generate further anti-idiotypic antibodies
(anti-anti-anti-idiotypic with respect to the original killer
toxin). The anti-anti-anti-idiotypic antibody of the invention can
in turn be used to generate further anti-idiotypic antibodies
(anti-anti-anti-anti-anti-idiotypic with respect to the original
killer toxin). Thus the microbicidal activity of the killer toxin
can be transferred through successive generations of anti-idiotypic
antibodies, and these various generations are within the scope of
the invention.
[0012] Thus the invention provides an antibody which recognises the
idiotope of an anti-idiotypic antibody of the invention (i.e. it
provides an anti-anti-anti-idiotypic antibody), an antibody which
recognises the idiotope of such an anti-anti-anti-idiotypic
antibody (i.e. an anti-anti-anti-anti-anti-idiotypic antibody) etc.
In general, therefore, the invention provides an antibody which
recognises the idiotope of an (anti-).sub.n-idiotypic antibody of
the invention, wherein n is an odd number (e.g. 1, 3, 5, 7, 9
etc.). These antibodies will generally bind to the idiotope of an
anti-toxin antibody such as KT4 and preferably have microbicidal
and/or antiviral activity.
[0013] For production of these (anti-).sub.n-idiotypic antibodies,
the invention provides (anti-).sub.m-idiotypic antibodies , wherein
m is an even number (e.g. 2, 4, 6, 8 etc.). These antibodies will
generally bind to a killer toxin.
[0014] The term `antibody` includes any of the various natural and
artificial antibodies and antibody-derived proteins which are
available. Thus the term `antibody` includes polyclonal antibodies,
monoclonal antibodies, Fab fragments, F(ab').sub.2 fragments, Fv
fragments, single-chain Fv (scFV) antibodies, oligobodies, etc.
[0015] To increase compatibility with the human immune system, it
is preferred to use human antibodies. As an alternative, antibodies
of the invention may be chimeric or humanized versions of non-human
antibodies [e.g. refs. 17 & 18 ].
[0016] In chimeric antibodies, non-human constant regions are
substituted by human constant regions but variable regions remain
non-human.
[0017] Humanized antibodies may be achieved by a variety of methods
including, for example: (1) grafting complementarity determining
regions (CDRs) from the non-human variable region onto a human
framework ("CDR-grafting"), with the optional additional transfer
of one or more framework residues from the non-human antibody
("humanizing"); (2) transplanting entire non-human variable
domains, but "cloaking" them with a human-like surface by
replacement of surface residues ("veneering "). In the present
invention, humanized antibodies include those obtained by
CDR-grafting, humanizing, and veneering or variable regions. [e.g.
refs. 19 to 25].
[0018] The constant regions of humanized antibodies are derived
from human immunoglobulins. The heavy chain constant region can be
selected from any of the five isotypes: .alpha., .delta.,
.epsilon., .gamma. or .mu..
[0019] Humanized or fully-human antibodies can also be produced
using transgenic animals that are engineered to contain human
immunoglobulin loci. For example, ref. 26 discloses transgenic
animals having a human lg locus wherein the animals do not produce
functional endogenous immunoglobulins due to the inactivation of
endogenous heavy and light chain loci. Ref. 27 also discloses
transgenic non-primate mammalian hosts capable of mounting an
immune response to an immunogen, wherein the antibodies have
primate constant and/or variable regions, and wherein the
endogenous immunoglobulin-encoding loci are substituted or
inactivated. Ref. 28 discloses the use of the Cre/Lox system to
modify the immunoglobulin locus in a mammal, such as to replace all
or a portion of the constant or variable region to form a modified
antibody molecule. Ref. 29 discloses non-human mammalian hosts
having inactivated endogenous lg loci and functional human Ig loci.
Ref. 30 discloses methods of making transgenic mice in which the
mice lack endogenous heavy chains, and express an exogenous
immunoglobulin locus comprising one or more xenogeneic constant
regions.
[0020] Antibodies of the invention may include a label. The label
may be detectable directly, such as a radioactive or fluorescent
label. Alternatively, the label may be detectable indirectly, such
as an enzyme whose products are detectable (e.g. luciferase,
.beta.-galactosidase, peroxidase etc.) or a binding partner such as
biotin and avidin or streptavidin.
[0021] Antibodies of the invention may be attached to a solid
support.
[0022] Antibodies of the invention may be produced by any suitable
means (e.g. by recombinant expression).
[0023] Preferred antibodies of the invention are single chain Fv
antibodies. These may be produced by joining heavy and light chain
variable regions from a starting monoclonal antibody of interest,
or may be identified by screening a scFv library (e.g. by phage
display). Reference 31 discloses a phage display method for
producing anti-idiotypic scFv antibodies.
[0024] Particularly preferred scFv antibodies of the invention are
H6 (SEQ IDs 1 & 2) and H20 (SEQ IDs 21 & 22). Antibodies
comprising one or more (e.g. 2, 3, 4, 5 or 6) of the CDRs from H6
and H20 are also preferred, as are derivatives of H6 and H20 in
which: (a) one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15) framework residues are substituted with other amino
acids; (b) the linker sequence (SEQ ID 30) is replaced with an
alternative linker sequence; (c) the `E-tag` sequence (SEQ ID 59)
is omitted or replaced. Fusion proteins comprising H6 or H20, or
derivatives (a) to (c), at the N- or C-terminus are also useful.
The H6 and H20 CDRs may optionally contain 1, 2, 3 or 4 amino acid
substitutions.
[0025] Other preferred antibodies of the invention are humanized
antibodies. Particularly preferred humanized antibodies of the
invention comprise one or more (e.g. 2, 3, 4, 5 or 6) CDRs from H6,
H20 or K20.
[0026] Polypeptides and Antibodyfragments
[0027] It has surprisingly been found that short fragments (e.g.
10mer fragments) of the variable regions of anti-idiotypic
antibodies of the invention can retain the antibodies' KT-like
microbicidal activity. Even more surprisingly, L-amino acids in
these fragments can be replaced with D-amino acids without removing
microbicidal activity, and amino acids within the fragments can be
substituted with other amino acids without removing microbicidal
activity. In addition, the fragments have been found to possess
anti-viral activity.
[0028] Thus the invention provides a polypeptide comprising: at
least one amino acid sequence which is a fragment of at least x
amino acids from the amino acid sequence of a variable region of an
antibody of the invention, optionally with y amino acid(s) within
said x amino acids being substituted by different amino acid(s).
The polypeptide preferably has microbicidal and/or antiviral
activity.
[0029] The polypeptide preferably consists of no more than 250
amino acids (e.g. no more than 225, 200, 190, 180, 170, 160, 150,
140, 130, 120, 110, 100, 95, 90, 80, 70, 60, 50, 45, 40, 35, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or even
5 amino acids). Polypeptides consisting of between 5 and 90 amino
acids are preferred (e.g. consisting of between 5 and 80, 5 and 70,
5 and 60 amino acids etc.). Particularly preferred are polypeptides
consisting of between 8 and 25 amino acids are preferred.
[0030] The polypeptide preferably consists of at least 3 amino
acids (e.g. at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100,
125, 150, 175, or at least 200 amino acids).
[0031] The value of x is preferably at least 3 (e.g. at least 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 45, 50, 55, 60, 70, 80, 90, 100, 125, 150, 175, or at least
200).
[0032] The value of y will be less than x and, depending on the
value of x, it may be x-1, x-2, x-3, x-4, x-5, x-6, x-7, x-8, x-9,
x-10, x- 11, x-12, x-13, x-14, or x-15. Preferred values of y are
1, 2, 3, 4 and 5. The y amino acids are typically substituted by A,
C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y. Each of
the y substitutions may be the same or different as the others. The
substitution is preferably by G or, more preferably, by A [32, 33].
The substituting amino acid may be an L- or a D- amino acid but,
where the other x amino acids all share a single
stereo-configuration (i.e. all D- or all L-), it preferably has
that stereo-configuration (although, of course, G has no
stereoisomers).
[0033] Where the fragment of x amino acids includes a C, the value
of y is preferably at least 1 such that the C is substituted for
another amino acid, such as S. Removal of C in this way improves
resistance to oxidation.
[0034] The fragment of at least x amino acids preferably includes
at least z amino acids from a CDR within the antibody. The value of
z is preferably at least 1 (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or
more).
[0035] The polypeptide may comprise more than one (e.g. 2, 3, 4, 5,
6) amino acid sequence each of which is a fragment of at least x
amino acids (e.g. SEQ ID 25). In such polypeptides, the value of x
in each fragment may be the same or different, the value of y
within each x may be the same or different, and the value of z
within each x may be the same or different. The fragments may be
joined by linker peptides such as glycine-rich linker sequences
(e.g. SEQ ID 30).
[0036] The invention also provides a polypeptide having formula
NH.sub.2--A--B--C--COOH, wherein: A is a polypeptide sequence
consisting of a amino acids; C is a polypeptide sequence consisting
of c amino acids; B is a polypeptide sequence which is, as defined
above, a fragment of at least x amino acids from the amino acid
sequence of a variable region of an antibody of the invention,
optionally with y amino acids within said x amino acids being
substituted by different amino acid(s). The polypeptide preferably
has microbicidal and/or antiviral activity.
[0037] The value of a is generally at least 1 (e.g. at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100, 150, 200, 250, 300, 350, 400, 450, 500 etc.). The value of c
is generally at least 1 (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10,
11,12, 13,14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250,
300, 350, 400, 450, 500 etc.). The value of a+c is at least 1 (e.g.
at least 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60,
70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 etc.). It
is preferred that the value of a+c is at most 1000 (e.g. at most
900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 190, 180,
170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30,
25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4,
3, 2).
[0038] The amino acid sequence of --A-- typically shares less than
m% sequence identity to the a amino acids which are N-terminal of
sequence --B-- in a variable region of an antibody of the invention
(e.g. in SEQ ID 2), and the amino acid sequence of --C-- typically
shares less than n% sequence identity to the c amino acids which
are C-terminal of sequence --B-- in a variable region of an
antibody of the invention (e.g. in SEQ ID 2). In general, the
values of m and n are both 60 or less (e.g. 50, 40, 30, 20, 10 or
less). The values of m and n may be the same as or different from
each other.
[0039] The polypeptide may comprise a mimotope of a yeast killer
toxin.
[0040] Preferred polypeptides comprise sequence
AA.sub.1-AA.sub.2AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.sub.7-AA.sub.8-AA-
.sub.9-AA.sub.10, wherein: each of AA.sub.1 . . . AA.sub.10 is
independently a D- or L- amino acid; AA.sub.1 is E, A or G;
AA.sub.2 is K, A or G; AA.sub.3 is V, A or G; AA.sub.4 is T, A or
G; AA.sub.5 is M, A or G; AA.sub.7 is T, A or G; AA.sub.7 is C, S,
A or G; AA.sub.8 is S, A or G; AA.sub.9 is A or G; and AA.sub.10 is
S, A or G; provided that no more than p of AA.sub.1, AA.sub.2,
AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.6, AA.sub.7, AA.sub.8,
AA.sub.9, and AA.sub.10 are A; and provided that no more than q of
AA.sub.1, AA.sub.2, AA.sub.3, AA.sub.4, AA.sub.5, AA.sub.6,
AA.sub.7, AA.sub.8, AA.sub.9, and AA.sub.10 are G. The value of p
is 1, 2, 3 or 4, and is preferably 1 or 2. The value of q is 0, 1
or 2 and is preferably 0 (i.e. no glycine residues) or 1.
[0041] Particularly preferred polypeptides comprise or consist of
amino acid sequences SEQ IDs : 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14,
15, 16, 17, 18, 19, 20, 23, 24, 25, 26, 27, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 &
71, with constituent amino acids in the D- and/or L- configuration.
SEQ IDs 3, 4, 23, 27 and 33 are most preferred.
[0042] For reference, the values of x, y and z for various of these
polypeptide sequences are as follows: TABLE-US-00001 SEQ ID x y z 3
10 0 3 4 10 1 3 5 10 1 3 6 10 1 3 7 10 1 3 8 10 1 3 9 10 1 3 10 10
1 3 11 10 1 3 12 10 1 3 14 9 1 2 15 8 1 1 16 7 1 0 17 6 1 0 18 5 1
0 19 4 1 0 20 3 1 0 23 10 2 3 24 10 0 3 26 15 0 8 27 9 0 8 32 10 2
3 33 10 2 3
Further microbicidal and/or antiviral fragments can be identified
by screening panels of overlapping peptide fragments once the
sequence of an anti-idiotypic antibody (e.g. H6, H20, K20) is known
e.g. by using the PepScan method [34]. Overlapping fragments of
4mers, 5mers, 6mers, 7mers, 8mers, 9mers, 10 mers, 11 mers, 12mers,
13mers etc. can be tested for microbicidal ability without
difficulty e.g. using the in vitro assays disclosed in the examples
herein.
[0043] Polypeptides of the invention may be linear, branched or
cyclic [35] but they are preferably linear chains of amino acids.
Where cysteine residues are present, polypeptides of the invention
may be linked to other polypeptides via disulfide bridges (and, in
particular, linked to other polypeptides of the invention to form
homodimers or heterodimers). Polypeptides of the invention may
comprise L-amino acids and/or D-amino acids. The inclusion of
D-amino acids is preferred in order to confer resistance to
mammalian proteases.
[0044] Polypeptide Production
[0045] Polypeptides of the invention may be produced by various
means.
[0046] A preferred method for production involves in vitro chemical
synthesis [36, 37]. Solid-phase peptide synthesis is particularly
preferred, such as methods based on t-Boc or Fmoc [38] chemistry.
Enzymatic synthesis [39] may also be used in part or in full.
[0047] As an alternative to chemical synthesis, biological
synthesis may be used e.g. the polypeptides may be produced by
translation. This may be carried out in vitro or in vivo.
Biological methods are in general restricted to the production of
polypeptides based on L-amino acids, but manipulation of
translation machinery (e.g. of aminoacyl-tRNA molecules) can be
used to allow the introduction of D-amino acids (or of other
non-natural amino acids, such as iodotyrosine or
methylphenylalanine, azidohomoalanine, etc.) [40]. Where D-amino
acids are included in the polypeptides of the invention, however,
it is preferred to use chemical synthesis. Further details on
polypeptide expression are given below.
[0048] To facilitate biological peptide synthesis, the invention
provides nucleic acid that encodes a polypeptide of the invention.
The invention also provides nucleic acid that encodes an antibody
of the invention.
[0049] The nucleic acid may be DNA or RNA (or hybrids thereof), or
their analogues, such as those containing modified backbones (e.g.
phosphorothioates) or peptide nucleic acids (PNA). It may be
single-stranded (e.g. mRNA) or double-stranded, and the invention
includes both individual strands of a double-stranded nucleic acid
(e.g. for antisense, priming or probing purposes). It may be linear
or circular. It may be labelled. It may be attached to a solid
support.
[0050] Nucleic acid according to the invention can, of course, be
prepared in many ways e.g. by chemical synthesis (e.g.
phosphoramidite synthesis of DNA) in whole or in part, by nuclease
digestion of longer molecules, by ligation of shorter molecules,
from genomic or cDNA libraries, by use of polymerases etc.
[0051] The invention provides vectors (e.g. plasmids) comprising
nucleic acid of the invention (e.g. expression vectors and cloning
vectors) and host cells (prokaryotic or eukaryotic) transformed
with such vectors.
[0052] These vectors can also be used for nucleic acid immunisation
[e.g. refs. 41, 42, 43, 44, 45 etc.]. Peptides can be expressed in
vivo in this way, as can therapeutic antibodies. It is also
possible to express idiotopes (e.g. the KT4 idiotope) to elicit
anti-idiotypic antibodies of the invention in a patient in
vivo.
[0053] Host cells which contain nucleic acid of the invention and
which express polypeptide or antibody of the invention may be used
as delivery vehicles e.g. commensal bacteria [46]. This is
particularly useful for delivery to mucosal surfaces.
[0054] The Yeast Killer Toxin
[0055] Killer toxins were originally identified in Saccharomyces
cerevisiae [47] and have since been identified in other genera of
yeasts including Pichia (such as P.anomala, P.kluyveri and
P.farinosa), Hanseniaspora (such as H.uvarum), Williopsis (such as
W.mrakii), Candida (such as C.maltosa), Debaryomyces (such as
D.hansenii), Schwanniomyces (such as S.occidentalis), Cryptococcus
(such as C.humicola), Torulopsis (such as T.glabrata), Ustilago
(such as U.maydis), Zygosaccharomyces (such as Z.bailii) and
Kluyveromyces (such as K.lactis and K.phaffii).
[0056] Any of these various toxins can be used with the present
invention. Preferred toxins are (a) those with a broad range of
microbicidal activity and (b) those from Pichia. A particularly
preferred toxin is the killer toxin from Pichia anomala
(`PaKT`).
[0057] Microbicidal Activity
[0058] The polypeptide or antibody of the invention preferably has
microbicidal activity.
[0059] Preferably, it has anti-mycotic activity and/or
anti-bacterial activity. Anti-bacterial activity may be against a
Gram-negative or Gram-positive bacterium.
[0060] More preferably, it has activity against a microbe which has
a glucan-based cell wall.
[0061] Microbes which are susceptible to polypeptides and
antibodies of the invention include bacteria, fungi and protozoa,
and include, but are not limited to: Candida species, such as
C.albicans; Cryptococcus species, such as C.neoformans;
Enterococcus species, such as E.faecalis; Streptococcus species,
such as S.pneumoniae, S.mutans, S.agalactiae and S.pyogenes;
Leishmania species, such as L.major and L.infantum; Acanthamoeba
species, such as A.castellani; Aspergillus species, such as
A.fumigatus; Pneumocystis species, such as P.carinii; Mycobacterium
species, such as M.tuberculosis; Pseudomonas species, such as
P.aeruginosa; Staphylococcus species, such as S.aureus; Salmonella
species, such as S.typhimurium; and Escherichia species, such as E.
coli.
[0062] Antiviral Activity
[0063] The polypeptide or antibody of the invention preferably has
antiviral activity.
[0064] Preferably, it has antiviral activity against a human virus,
such as a myxovirus (e.g. an orthomyxovirus) or a retrovirus (e.g.
a lentivirus).
[0065] Viruses which are susceptible to polypeptides and antibodies
of the invention include, but are not limited to: influenza virus
(A or B), human immunodeficiency virus (HIV-1, HIV-2, HIV-O),
respiratory syncytial virus (RSV), yellow fever virus, etc.
[0066] Drug Design and Peptidomimetics
[0067] Polypeptides of the invention are useful microbicides and
antivirals in their own right. However, they may be refined to
improve microbicidal and/or antiviral activity (either general or
specific) or to improve pharmacologically important features such
as bio-availability, toxicology, metabolism, pharmacokinetics etc.
The polypeptides may therefore be used as lead compounds for
further research and refinement.
[0068] Polypeptides of the invention can be used for designing
peptidomimetic molecules [e.g. refs. 48 to 53] with microbicidal
and/or antiviral activity. These will typically be isosteric with
respect to the polypeptides of the invention but will lack one or
more of their peptide bonds. For example, the peptide backbone may
be replaced by a non-peptide backbone while retaining important
amino acid side chains.
[0069] The peptidomimetic molecule may comprise sugar amino acids
[54]. Peptoids may be used.
[0070] To assist in the design of peptidomimetic molecules, a
pharmacophore (i.e. a collection of chemical features and 3D
constraints that expresses specific characteristics responsible for
activity) can be defined for the KM peptides. The pharmacophore
preferably includes surface-accessible features, more preferably
including hydrogen bond donors and acceptors, charged/ionisable
groups, and/or hydrophobic patches. These may be weighted depending
on their relative importance in conferring activity [55].
[0071] Pharmacophores can be determined using software such as
CATALYST (including HypoGen or HipHop) [56], CERIUS.sup.2, or
constructed by hand from a known conformation of a polypeptide of
the invention. The pharmacophore can be used to screen structural
libraries, using a program such as CATALYST. The CLIX program [57]
can also be used, which searches for orientations of candidate
molecules in structural databases that yield maximum spatial
coincidence with chemical groups which interact with the
receptor.
[0072] The binding surface or pharmacophore can be used to map
favourable interaction positions for functional groups (e.g.
protons, hydroxyl groups, amine groups, hydrophobic groups) or
small molecule fragments. Compounds can then be designed de novo in
which the relevant functional groups are located in substantially
the same spatial relationship as in polypeptides of the
invention.
[0073] Functional groups can be linked in a single compound using
either bridging fragments with the correct size and geometry or
frameworks which can support the functional groups at favourable
orientations, thereby providing a peptidomimetic compound according
to the invention. Whilst linking of functional groups in this way
can be done manually, perhaps with the help of software such as
QUANTA or SYBYL, automated or semi-automated de novo design
approaches are also available, such as: [0074] MCSS/HOOK [58, 59,
56], which links multiple functional groups with molecular
templates taken from a database. [0075] LUDI [60, 56], which
computes the points of interaction that would ideally be fulfilled
by a ligand, places fragments in the binding site based on their
ability to interact with the receptor, and then connects them to
produce a ligand. [0076] MCDLNG [61], which fills a receptor
binding site with a close-packed array of generic atoms and uses a
Monte Carlo procedure to randomly vary atom types, positions,
bonding arrangements and other properties. [0077] GROW [62], which
starts with an initial `seed` fragment (placed manually or
automatically) and grows the ligand outwards. [0078] SPROUT [63],
suite which includes modules to: identify favourable hydrogen
bonding and hydrophobic regions within a binding pocket (HIPPO
module); select functional groups and position them at target sites
to form starting fragments for structure generation (EleFAnT);
generate skeletons that satisfy the steric constraints of the
binding pocket by growing spacer fragments onto the start fragments
and then connecting the resulting part skeletons (SPIDeR);
substitute hetero atoms into the skeletons to generate molecules
with the electrostatic properties that are complementary to those
of the receptor site (MARABOU). The solutions can be clustered and
scored using the ALLigaTOR module. [0079] CAVEAT [64], which
designs linking units to constrain acyclic molecules. [0080]
LEAPFROG [65], which evaluates ligands by making small stepwise
structural changes and rapidly evaluating the binding energy of the
new compound. Changes are kept or discarded based on the altered
binding energy, and structures evolve to increase the interaction
energy with the receptor. [0081] GROUPBUILD [66], which uses a
library of common organic templates and a complete empirical force
field description of the non-bonding interactions between a ligand
and receptor to construct ligands that have chemically reasonable
structure and have steric and electrostatic properties
complimentary to the receptor binding site. [0082] RASSE [67]
[0083] These methods identify microbicidal compounds. These
compounds may be designed de novo, may be known compounds, or may
be based on known compounds. The compounds may be useful
microbicides and/or antivirals themselves, or they may be
prototypes which can be used for further pharmaceutical refinement
(i.e. lead compounds) in order to improve binding affinity or other
pharmacologically important features (e.g. bio-availability,
toxicology, metabolism, pharmacokinetics etc.).
[0084] The invention thus provides: (i) a compound identified using
these drug design methods; (ii) a compound identified using these
drug design methods, for use as a pharmaceutical; (iii) the use of
a compound identified using these drug design methods in the
manufacture of a microbicide and/or an antiviral; and (iv) a method
of treating a patient with a microbial or viral infection,
comprising administering an effective amount of a compound
identified using these drug design methods.
[0085] As well as being useful compounds individually, ligands
identified in silico by the structure-based design techniques can
also be used to suggest libraries of compounds for `traditional` in
vitro or in vivo screening methods. Important pharmaceutical motifs
in the ligands can be identified and mimicked in compound libraries
(e.g. combinatorial libraries) for screening for microbicidal
and/or antiviral activity.
[0086] Pharmaceutical Compositions
[0087] The invention provides a pharmaceutical composition
comprising (a) polypeptide, peptidomimetic, nucleic acid and/or
antibody of the invention and (b) a pharmaceutical carrier.
[0088] Component (a) is the active ingredient in the composition,
and this is present at a therapeutically effective amount i.e. an
amount sufficient to inhibit microbial/viral growth and/or survival
in a patient, and preferably an amount sufficient to eliminate
microbial infection. The precise effective amount for a given
patient will depend upon their size and health, the nature and
extent of infection, and the composition or combination of
compositions selected for administration. The effective amount can
be determined by routine experimentation and is within the judgment
of the clinician. For purposes of the present invention, an
effective dose will generally be from about 0.01 mg/kg to about 5
mg/kg, or about 0.01 mg/ kg to about 50 mg/kg or about 0.05 mg/kg
to about 10 mg/kg. Pharmaceutical compositions based on
polypeptides, antibodies and nucleic acids are well known in the
art. Polypeptides may be included in the composition in the form of
salts and/or esters.
[0089] Carrier (b) can be any substance that does not itself induce
the production of antibodies harmful to the patient receiving the
composition, and which can be administered without undue toxicity.
Suitable carriers can be large, slowly metabolized macromolecules
such as proteins, polysaccharides, polylactic acids, polyglycolic
acids, polymeric amino acids, amino acid copolymers, and inactive
virus particles. Such carriers are well known to those of ordinary
skill in the art. Pharmaceutically acceptable carriers can include
liquids such as water, saline, glycerol and ethanol. Auxiliary
substances, such as wetting or emulsifying agents, pH buffering
substances, and the like, can also be present in such vehicles.
Liposomes are suitable carriers. A thorough discussion of
pharmaceutical carriers is available in ref. 68.
[0090] Viral and microbial infections affect various areas of the
body and so the compositions of the invention may be prepared in
various forms. For example, the compositions may be prepared as
injectables, either as liquid solutions or suspensions. Solid forms
suitable for solution in, or suspension in, liquid vehicles prior
to injection can also be prepared. The composition may be prepared
for topical administration e.g. as an ointment, cream or powder.
The composition be prepared for oral administration e.g. as a
tablet or capsule, or as a syrup (optionally flavoured). The
composition may be prepared for pulmonary administration e.g. as an
inhaler, using a fine powder or a spray. The composition may be
prepared as a suppository or pessary. The composition may be
prepared for nasal, aural or ocular administration e.g. as drops,
as a spray, or as a powder [e.g. 69]. The composition may be
included in a mouthwash. The composition may be lyophilised.
[0091] The pharmaceutical composition is preferably sterile. It is
preferably pyrogen-free. It is preferably buffered e.g. at between
pH 6 and pH 8, generally around pH 7.
[0092] The invention also provides a delivery device containing a
pharmaceutical composition of the invention. The device may be, for
example, a syringe or an inhaler.
[0093] Compositions of the invention may be used in conjunction
with known anti-fungals. Suitable anti-fungals include, but are not
limited to, azoles (e.g. fluconazole, itraconazole), polyenes (e.g.
amphotericin B), flucytosine, and squalene epoxidase inhibitors
(e.g. terbinafine) [see also ref. 70]. Compositions may also be
used in conjunction with known antivirals e.g. HIV protease
inhibitors, a 2',3'-dideoxynucleoside (e.g. DDC, DDI),
3'-azido-2',3'-dideoxynucleosides (AZT),
3'-fluoro-2',3'-dideoxynucleosides (FLT),
2',3'-didehydro-2',3'-dideoxynucleosides (e.g. D4C, D4T) and
carbocyclic derivatives thereof (e.g. carbovir),
2'-fluoro-ara-2',3'-dideoxynucleosides, 1,3-dioxolane derivatives
(e.g. 2',3'-dideoxyl-3'-thiacytidine), oxetanocin analogues and
carbocyclic derivatives thereof (e.g. cyclobut-G) and the
9-(2-phosphonylmethoxyethyl)adenine (PMEA) and
9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives,
tetrahydro-imidazo[4,5,l -jk][1,4]-benzodiazepin-2(1H)one (TIBO),
1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT),
dipyrido[3,2-b:2',3'-e]-[1,4]diazepin-6-one (nevirapine) and
pyridin-2(1H)one derivatives, 3TC, etc.
[0094] Medical Treatments and Uses
[0095] The invention provides antibody, polypeptide, peptidomimetic
or nucleic acid of the invention for use as a medicament. The
invention also provides a method for treating a patient suffering
from a microbial and/or viral infection, comprising administering
to the patient a pharmaceutical composition of the invention. The
invention also provides the use of antibody, polypeptide,
peptidomimetic or nucleic acid of the invention in the manufacture
of a medicament for treating a patient.
[0096] The patient is preferably a human. The human may be an adult
or, preferably, a child. The human may be immunocompromised.
[0097] These uses and methods are particularly useful for treating
infections of: Candida species, such as C.albicans; Cryptococcus
species, such as C.neoformans; Enterococcus species, such as
E.faecalis; Streptococcus species, such as S.pneumoniae, S.mutans,
S.agalactiae and S.pyogenes; Leishmania species, such as L.major
and L.infantum; Acanthamoeba species, such as A.castellani;
Aspergillus species, such as A.fumigatus and A.flavus; Pneumocystis
species, such as P.carinii; Mycobacterium species, such as
M.tuberculosis; Pseudomonas species, such as P.aeruginosa;
Staphylococcus species, such as S.aureus; Salmonella species, such
as S.typhimurium; Coccidioides species such as C.immitis;
Trichophyton species such as Tverrucosum; Blastomyces species such
as B.dermatidis; Histoplasma species such as H.capsulatum;
Paracoccidioides species such as P.brasiliensis; Pythiumn species
such as P.insidiosum; and Escherichia species, such as E.coli. They
are also useful for treating infections of: influenza viruses and
HIV.
[0098] The uses and methods are particularly useful for treating
diseases including, but not limited to: candidosis, aspergillosis,
cryptococcosis, dermatomycoses, sporothrychosis and other
subcutaneous mycoses, blastomycosis, histoplasmosis,
coccidiomycosis, paracoccidiomycosis, pneumocystosis, thrush,
tuberculosis, mycobacteriosis, respiratory infections, scarlet
fever, pneumonia, impetigo, rheumatic fever, sepsis, septicaemia,
cutaneous and visceral leishmaniasis, comeal acanthamoebiasis,
keratitis, cystic fibrosis, typhoid fever, gastroenteritis and
hemolytic-uremic syndrome, flu and AIDS. Anti-C.albicans activity
is particularly useful for treating infections in AIDS
patients.
[0099] Efficacy of treatment can be tested by monitoring
microbial/viral infection after administration of the
pharmaceutical composition of the invention.
[0100] Compositions of the invention will generally be administered
directly to a patient. Direct delivery may be accomplished by
parenteral injection (e.g. subcutaneously, intraperitoneally,
intravenously, intramuscularly, or to the interstitial space of a
tissue), or by rectal, oral, vaginal, topical, transdermal, ocular,
nasal, aural, or pulmonary administration. Injection or intranasal
administration is preferred.
[0101] Dosage treatment can be a single dose schedule or a multiple
dose schedule.
[0102] Pharmaceutical compositions of the invention may also be
used prophylactically e.g. in a situation where contact with
microbes is expected and where establishment of infection is to be
prevented. For instance, the composition may be administered prior
to surgery.
[0103] Polypeptide Expression and Other General Techniques
[0104] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, recombinant DNA, and immunology, which are within the
skill of the art. Such techniques are explained fully in the
literature e.g. Sambrook Molecular Cloning; A Laboratory Manual,
Second Edition (1989); DNA Cloning, Volumes I and ii (D. N Glover
ed. 1985); Oligonucleolide Synthesis (M. J. Gait ed, 1984); Nucleic
Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984);
Transcription and Translation (B. D. Hames & S. J. Higgins eds.
1984); Animal Cell Culture (R. I. Freshney ed. 1986); Immobilized
Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide
to Molecular Cloning (1984); the Methods in Enzymology series
(Academic Press, Inc.), especially volumes 154 & 155; Gene
Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos
eds. 1987, Cold Spring Harbor Laboratory); Mayer and Walker, eds.
(1987), Immunochemical Methods in Cell and Molecular Biology
(Academic Press, London); Scopes, (1987) Protein Purification:
Principles and Practice, Second Edition (Springer-Verlag, N.Y.),
and Handbook of Experimental Immunology, Volumes I-IV (Weir&
Blackwell eds 1986).
[0105] Standard abbreviations for nucleotides and amino acids are
used in this specification: TABLE-US-00002 Nucleotides A Adenine C
Cytosine G Guanine T Thymine U Uracil
[0106] TABLE-US-00003 Degenerate nucleotide codes in addition to
the five above codes: N any R puRine Y pYrimidine K Keto (A/C/G/T)
(G/A) (T/C) (G/T) M aMino S Strong W Weak B not A (A/C) (G/C) (A/T)
(C/G/T) D not C H not G V not T (A/G/T) (A/C/T) (A/C/G)
[0107] TABLE-US-00004 Amino acids A Alanine C Cysteine D Aspartate
E Glutamate F Phenyl- G Glycine H Histidine I Isoleucine alanine L
Leucine M Methionine N Asparagine K Lysine Q Glutamine R Arginine S
Serine P Proline V Valine W Tryptophan Y Tyrosine T Threonine
The invention provides a polypeptide or nucleic acid comprising or
consisting of any one of amino acid or nucleotide sequences given
in the sequence listing.
[0108] Definitions
[0109] The term "comprising" means "including" as well as
"consisting" e.g. a composition "comprising" X may consist
exclusively of X or may include something additional to X, such as
X+Y.
[0110] A composition containing X is "substantially free of" Y when
at least 85% by weight of the total X+Y in the composition is X.
Preferably, X comprises at least about 90% by weight of the total
of X+Y in the composition, more preferably at least about 95% or
even 99% by weight. The term "heterologous" refers to two
biological components that are not found together in nature. The
components may be host cells, genes, or regulatory regions, such as
promoters. Although the heterologous components are not found
together in nature, they can function together, as when a promoter
heterologous to a gene is operably linked to the gene. Another
example is where an influenza sequence is heterologous to a mouse
host cell. Further examples would be two epitopes from the same or
different proteins which have been assembled in a single protein in
an arrangement not found in nature.
[0111] An "origin of replication" is a polynucleotide sequence that
initiates and regulates replication of polynucleotides, such as an
expression vector. The origin of replication behaves as an
autonomous unit of polynucleotide replication within a cell,
capable of replication under its own control. An origin of
replication may be needed for a vector to replicate in a particular
host cell. With certain origins of replication, an expression
vector can be reproduced at a high copy number in the presence of
the appropriate proteins within the cell. Examples of origins are
the autonomously replicating sequences, which are effective in
yeast; and the viral T-antigen, effective in COS-7 cells. A
"mutant" sequence is defined as DNA, RNA or amino acid sequence
differing from but having sequence identity with the native or
disclosed sequence. Depending on the particular sequence, the
degree of sequence identity between the native or disclosed
sequence and the mutant sequence is preferably greater than 50%
(e.g. 60%, 70%, 80%, 90%, 95%, 99% or more, calculated using the
Smith-Waterman algorithm as described above). As used herein, an
"allelic variant" of a nucleic acid molecule, or region, for which
nucleic acid sequence is provided herein is a nucleic acid
molecule, or region, that occurs essentially at the same locus in
the genome of another or second isolate, and that, due to natural
variation caused by, for example, mutation or recombination, has a
similar but not identical nucleic acid sequence. A coding region
allelic variant typically encodes a protein having similar activity
to that of the protein encoded by the gene to which it is being
compared. An allelic variant can also comprise an alteration in the
5' or 3' untranslated regions of the gene, such as in regulatory
control regions (e.g. see U.S. Pat. No. 5,753,235).
[0112] Polypeptide Expression
[0113] Nucleotide sequences can be expressed in a variety of
different expression systems; for example those used with mammalian
cells, baculoviruses, plants, bacteria, and yeast.
[0114] Generally, any system or vector that is suitable to
maintain, propagate or express nucleic acid molecules to produce a
polypeptide in the required host may be used. The appropriate
nucleotide sequence may be inserted into an expression system by
any of a variety of well-known and routine techniques, such as, for
example, those described in Sambrook et. al. Generally, the
encoding gene can be placed under the control of a control element
such as a promoter, ribosome binding site (for bacterial
expression) and, optionally, an operator, so that the DNA sequence
encoding the desired polypeptide is transcribed into RNA in the
transformed host cell.
[0115] Examples of suitable expression systems include, for
example, chromosomal, episomal and virus-derived systems,
including, for example, vectors derived from: bacterial plasmids,
bacteriophage, transposons, yeast episomes, insertion elements,
yeast chromosomal elements, viruses such as baculoviruses, papova
viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox
viruses, pseudorabies viruses and retroviruses, or combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, including cosmids and phagemids. Human artificial
chromosomes (HACs) may also be employed to deliver larger fragments
of DNA than can be contained and expressed in a plasmid.
[0116] Particularly suitable expression systems include
microorganisms such as bacteria transformed with recombinant
bacteriophage, plasmid or cosmid DNA expression vectors; yeast
transformed with yeast expression vectors; insect cell systems
infected with virus expression vectors (for example, baculovirus);
plant cell systems transformed with virus expression vectors (for
example, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
or with bacterial expression vectors (for example, Ti or pBR322
plasmids); or animal cell systems. Cell-free translation systems
can also be employed to produce the polypeptides of the
invention.
[0117] Introduction of nucleic acid molecules encoding a
polypeptide of the present invention into host cells can be
effected by methods described in many standard laboratory manuals,
such as Davis et al., Basic Methods in Molecular Biology (1986) and
Sambrook et al., (supra). Particularly suitable methods include
calcium phosphate transfection, DEAE-dextran mediated transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation, transduction, scrape loading, ballistic
introduction or infection (see Sambrook et al., 1989 [supra];
"Current Protocols in Molecular Biology", Ausubel et al. (eds).
Greene Publishing Association and John Wiley lnterscience, New
York, 1989, 1992; Spector, Goldman & Leinwald, 1998). In
eukaryotic cells, expression systems may either be transient (for
example, episomal) or permanent (chromosomal integration) according
to the needs of the system.
[0118] For long-term, high-yield production of a recombinant
polypeptide, stable expression is preferred. For example, cell
lines which stably express the polypeptide of interest may be
transformed using expression vectors which may contain viral
origins of replication and/or endogenous expression elements and a
selectable marker gene on the same or on a separate vector.
Following the introduction of the vector, cells may bc allowed to
grow for 1-2 days in an enriched media before they are switched to
selective media. The purpose of the selectable marker is to confer
resistance to selection, and its presence allows growth and
recovery of cells that successfully express the introduced
sequences. Resistant clones of stably transformed cells may be
proliferated using tissue culture techniques appropriate to the
cell type.
[0119] i. Mammalian Systems
[0120] Mammalian expression systems are known in the art. A
mammalian promoter is any DNA sequence capable of binding mammalian
RNA polymerase and initiating the downstream (3') transcription of
a coding sequence (e.g. structural gene) into mRNA. A promoter will
have a transcription initiating region, which is usually placed
proximal to the 5' end of the coding sequence, and a TATA box,
usually located 25-30 base pairs (bp) upstream of the transcription
initiation site. The TATA box is thought to direct RNA polymerase
II to begin RNA synthesis at the correct site. A mammalian promoter
will also contain an upstream promoter element, usually located
within 100 to 200 bp upstream of the TATA box. An upstream promoter
element determines the rate at which transcription is initiated and
can act in either orientation [Sambrook et al. (1989) "Expression
of Cloned Genes in Mammalian Cells." In Molecular Cloning: A
Laboratory Manual, 2nd ed.].
[0121] Mammalian viral genes are often highly expressed and have a
broad host range; therefore sequences encoding mammalian viral
genes provide particularly useful promoter sequences. Examples
include the SV40 early promoter, mouse mammary tumor virus LTR
promoter, adenovirus major late promoter (Ad MLP), and herpes
simplex virus promoter. In addition, sequences derived from
non-viral genes, such as the murine metallotheionein gene, also
provide useful promoter sequences. Expression may be either
constitutive or regulated (inducible), depending on the promoter
can be induced with glucocorticoid in hormone-responsive cells.
[0122] The presence of an enhancer element (enhancer), combined
with the promoter elements described above, will usually increase
expression levels. An enhancer is a regulatory DNA sequence that
can stimulate transcription up to 1000-fold when linked to
homologous or heterologous promoters, with synthesis beginning at
the normal RNA start site. Enhancers are also active when they are
placed upstream or downstream from the transcription initiation
site, in either normal or flipped orientation, or at a distance of
more than 1000 nucleotides from the promoter [Maniatis et al.
(1987) Science 236:1237; Alberts et al. (1989) Molecular Biology of
the Cell, 2nd ed.]. Enhancer elements derived from viruses may be
particularly useful, because they usually have a broader host
range. Examples include the SV40 early gene enhancer [Dijkema et al
(1985) EMBO J. 4:761 ] and the enhancer/promoters derived from the
long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al.
(1982b) PNAS USA 79:6777] and from human cytomegalovirus [Boshart
et al. (1985) Cell 41:521]. Additionally, some enhancers are
regulatable and become active only in the presence of an inducer,
such as a hormone or metal ion [Sassone-Corsi and Borelli (1986)
Trends Genel. 2:215; Maniatis et al. (1987) Science 236:1237].
[0123] A DNA molecule may be expressed intracellularly in mammalian
cells. A promoter sequence may be directly linked with the DNA
molecule, in which case the first amino acid at the N-terminus of
the recombinant protein will always be a methionine, which is
encoded by the ATG start codon. If desired, the N-terminus may be
cleaved by in vitro incubation with cyanogen bromide.
[0124] Alternatively, foreign proteins can also be secreted from
the cell into the growth media by creating chimeric DNA molecules
that encode a fusion protein comprised of a leader sequence
fragment that provides for secretion of the foreign protein in
mammalian cells. Preferably, there are processing sites encoded
between the leader fragment and the foreign gene that can be
cleaved either in vivo or in vitro. The leader sequence fragment
usually encodes a signal peptide comprised of hydrophobic amino
acids which direct the secretion of the protein from the cell. The
adenovirus triparite leader is an example of a leader sequence that
provides for secretion of a foreign protein in mammalian cells.
[0125] Usually, transcription termination and polyadenylation
sequences recognized by mammalian cells are regulatory regions
located 3' to the translation stop codon and thus, together with
the promoter elements, flank the coding sequence. The 3' terminus
of the mature mRNA is formed by site-specific post-transcriptional
cleavage and polyadenylation [Birnstiel et al. (1985) Cell 41:349;
Proudfoot and Whitelaw (1988) "Termination and 3' end processing of
eukaryotic RNA. In Transcription and splicing (ed. B. D. Hames and
D. M. Glover); Proudfoot (1989) Trends Biochem. Sci. 14:105]. These
sequences direct the transcription of an mRNA which can be
translated into the polypeptide encoded by the DNA. Examples of
transcription terminater/polyadenylation signals include those
derived from SV40 [Sambrook et al (1989) "Expression of cloned
genes in cultured mammalian cells." In Molecular Cloning: A
Laboratory Manual].
[0126] Usually, the above described components, comprising a
promoter, polyadenylation signal, and transcription termination
sequence are put together into expression constructs. Enhancers,
introns with functional splice donor and acceptor sites, and leader
sequences may also be included in an expression construct, if
desired. Expression constructs are often maintained in a replicon,
such as an extrachromosomal element (e.g. plasmids) capable of
stable maintenance in a host, such as mammalian cells or bacteria.
Mammalian replication systems include those derived from animal
viruses, which require trans-acting factors to replicate. For
example, plasmids containing the replication systems of
papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or
polyomavirus, replicate to extremely high copy number in the
presence of the appropriate viral T antigen. Additional examples of
mammalian replicons include those derived from bovine
papillomavirus and Epstein-Barr virus. Additionally, the replicon
may have two replicaton systems, thus allowing it to be maintained,
for example, in mammalian cells for expression and in a prokaryotic
host for cloning and amplification. Examples of such
mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al.
(1989) Mol. Cell. Biol. 9:946] and pHEBO [Shimizu et al. (1986) Mol
Cell. Biol. 6:1074].
[0127] The transformation procedure used depends upon the host to
be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are known in the art and
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0128] Mammalian cell lines available as hosts for expression are
known in the art and include many immortalised cell lines available
from the American Type Culture Collection (ATCC) including, but not
limited to, Chinese hamster ovary (CHO), HeLa, baby hamster kidney
(BHK), monkey kidney (COS), C127, 3T3, BHK, HEK 293, Bowes melanoma
and human hepatocellular carcinoma (for example Hep G2) cells and a
number of other cell lines.
[0129] ii. Baculovirus Systems
[0130] The polynucleotide encoding the protein can also be inserted
into a suitable insect expression vector, and is operably linked to
the control elements within that vector. Vector construction
employs techniques which are known in the art. Generally, the
components of the expression system include a transfer vector,
usually a bacterial plasmid, which contains both a fragment of the
baculovirus genome, and a convenient restriction site for insertion
of the heterologous gene or genes to be expressed; a wild type
baculovirus with a sequence homologous to the baculovirus-specific
fragment in the transfer vector (this allows for the homologous
recombination of the heterologous gene in to the baculovirus
genome); and appropriate insect host cells and growth media.
[0131] After inserting the DNA sequence encoding the protein into
the transfer vector, the vector and the wild type viral genome are
transfected into an insect host cell where the vector and viral
genome are allowed to recombine. The packaged recombinant virus is
expressed and recombinant plaques are identified and purified.
Materials and methods for baculovirus/insect cell expression
systems are commercially available in kit form e.g. from
Invitrogen, San Diego Calif. ("MaxBac" kit). These techniques are
generally known to those skilled in the art and fully described in
Summers & Smith, Texas Agricultural Experiment Station Bulletin
No. 1555 (1987) (hereinafter "Summers & Smith").
[0132] Prior to inserting the DNA sequence encoding the protein
into the baculovirus genome, the above described components,
comprising a promoter, leader (if desired), coding sequence of
interest, and transcription termination sequence, are usually
assembled into an intermediate transplacement construct (transfer
vector). This construct may contain a single gene and operably
linked regulatory elements; multiple genes, each with its owned set
of operably linked regulatory elements; or multiple genes,
regulated by the same set of regulatory elements. Intermediate
transplacement constructs are often maintained in a replicon, such
as an extrachromosomal element (e.g. plasmids) capable of stable
maintenance in a host, such as a bacterium. The replicon will have
a replication system, thus allowing it to be maintained in a
suitable host for cloning and amplification.
[0133] Currently, the most commonly used transfer vector for
introducing foreign genes into AcNPV is pAc373. Many other vectors,
known to those of skill in the art, have also been designed. These
include, for example, pVL985 (which alters the polyhedrin start
codon from ATG to ATT, and which introduces a BamHI cloning site 32
basepairs downstream from the ATT; see Luckow and Summers, Virology
(1989) 17:31.
[0134] The plasmid usually also contains a polyhedrin
polyadenylation signal (Miller (1988) Ann. Rev. Microbiol. 42:177)
and a prokaryotic ampicillin-resistance (amp) gene and origin of
replication for selection and propagation in E.coli.
[0135] Baculovirus transfer vectors usually contain a baculovirus
promoter. A baculovirus promoter is any DNA sequence capable of
binding a baculovirus RNA polymerase and initiating the downstream
(5' to 3') transcription of a coding sequence (e.g. structural
gene) into mRNA. A promoter will have a transcription initiation
region which is usually placed proximal to the 5' end of the coding
sequence. This transcription initiation region usually includes an
RNA polymerase binding site and a transcription initiation site. A
baculovirus transfer vector may also have a second domain called an
enhancer, which, if present, is usually distal to the structural
gene. Expression may be either regulated or constitutive.
[0136] Structural genes, abundantly transcribed at late times in a
viral infection cycle, provide particularly useful promoter
sequences. Examples include sequences derived from the gene
encoding the viral polyhedron protein, Friesen et al., (1986) "The
Regulation of Baculovirus Gene Expression," in The Molecular
Biology of Baculoviruses (ed. Walter Doerfler); EP-127839 &
EP-155476; and the gene encoding the p10 protein, Vlak et al.
(1988), J Gen. ViroL 69:765.
[0137] DNA encoding suitable signal sequences can be derived from
genes for secreted insect or baculovirus proteins, such as the
baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409).
Alternatively, since the signals for mammalian cell
posttranslational modifications (such as signal peptide cleavage,
proteolytic cleavage, and phosphorylation) appear to be recognized
by insect cells, and the signals required for secretion and nuclear
accumulation also appear to be conserved between the invertebrate
cells and vertebrate cells, leaders of non-insect origin, such as
those derived from genes encoding human .quadrature.-inteferon,
Maeda et al., (1985), Nature 315:592; human gastrin-releasing
peptide, Lebacq-Verheyden et al., (1988), Molec. Cell Biol. 8:3129;
human IL-2, Smith et al., (1985) Proc. Nat'l Acad Sci. USA,
82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and
human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also
be used to provide for secretion in insects.
[0138] A recombinant polypeptide or polyprotein may be expressed
intracellularly or, if it is expressed with the proper regulatory
sequences, it can be secreted. Good intracellular expression of
nonfused foreign proteins usually requires heterologous genes that
ideally have a short leader sequence containing suitable
translation initiation signals preceding an ATG start signal. If
desired, methionine at the N-terminus may be cleaved from the
mature protein by in vitro incubation with cyanogen bromide.
[0139] Alternatively, recombinant polyproteins or proteins which
are not naturally secreted can be secreted from the insect cell by
creating chimeric DNA molecules that encode a fusion protein
comprised of a leader sequence fragment that provides for secretion
of the foreign protein in insects. The leader sequence fragment
usually encodes a signal peptide comprised of hydrophobic amino
acids which direct the translocation of the protein into the
endoplasmic reticulum.
[0140] After insertion of the DNA sequence and/or the gene encoding
the expression product precursor of the protein, an insect cell
host is co-transformed with the heterologous DNA of the transfer
vector and the genomic DNA of wild type baculovirus--usually by
co-transfection. The promoter and transcription termination
sequence of the construct will usually comprise a 2-5kb section of
the baculovirus genome. Methods for introducing heterologous DNA
into the desired site in the baculovirus virus are known in the
art. (See Summers & Smith supra; Ju et al. (1987); Smith et
al., Mol. Cell Biol. (1983) 3:2156; and Luckow and Summers (1989)).
For example, the insertion can be into a gene such as the
polyhedrin gene, by homologous double crossover recombination;
insertion can also be into a restriction enzyme site engineered
into the desired baculovirus gene. Miller et al., (1989), Bioessoys
4:91.The DNA sequence, when cloned in place of the polyhedrin gene
in the expression vector, is flanked both 5' and 3' by
polyhedrin-specific sequences and is positioned downstream of the
polyhedrin promoter.
[0141] The newly formed baculovirus expression vector is
subsequently packaged into an infectious recombinant baculovirus.
Homologous recombination occurs at low frequency (between about 1%
and about 5%); thus, the majority of the virus produced after
cotransfection is still wild-type virus. Therefore, a method is
necessary to identify recombinant viruses. An advantage of the
expression system is a visual screen allowing recombinant viruses
to be distinguished. The polyhedrin protein, which is produced by
the native virus, is produced at very high levels in the nuclei of
infected cells at late times after viral infection. Accumulated
polyhedrin protein forms occlusion bodies that also contain
embedded particles. These occlusion bodies, up to 15 .quadrature.m
in size, are highly refractile, giving them a bright shiny
appearance that is readily visualized under the light microscope.
Cells infected with recombinant viruses lack occlusion bodies. To
distinguish recombinant virus from wild-type virus, the
transfection supernatant is plaqued onto a monolayer of insect
cells by techniques known to those skilled in the art. Namely, the
plaques are screened under the light microscope for the presence
(indicative of wild-type virus) or absence (indicative of
recombinant virus) of occlusion bodies. Current Protocols in
Microbiology Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10,1990);
Summers & Smith, supra; Miller et al. (1989). Recombinant
baculovirus expression vectors have been developed for infection
into several insect cells. For example, recombinant baculoviruses
have been developed for, inter alia: Aedes aegypli, Aulographa
californica, Bombyx mori, Drosophila melanogaster,
Spodoplerafrugiperda, and Trichoplusia ni (WO 89/046699; Carbonell
et al., (1985) J ViroL 56:153; Wright (1986) Nature 321:718; Smith
et al., (1983) Mol. CelL BioL 3:2156; and see generally, Fraser, et
al. (1989) In Vitro Cell. Dev. Biol. 25:225).
[0142] Cells and cell culture media are commercially available for
both direct and fusion expression of heterologous polypeptides in a
baculovirus/expression system; cell culture technology is generally
known to those skilled in the art. See, e.g. Summers & Smith
supra.
[0143] The modified insect cells may then be grown in an
appropriate nutrient medium, which allows for stable maintenance of
the plasmid(s) present in the modified insect host. Where the
expression product gene is under inducible control, the host may be
grown to high density, and expression induced. Alternatively, where
expression is constitutive, the product will be continuously
expressed into the medium and the nutrient medium must be
continuously circulated, while removing the product of interest and
augmenting depleted nutrients. The product may be purified by such
techniques as chromatography, e.g. HPLC, affinity chromatography,
ion exchange chromatography, etc.; electrophoresis; density
gradient centrifugation; solvent extraction, or the like. As
appropriate, the product may be further purified, as required, so
as to remove substantially any insect proteins which are also
secreted in the medium or result from lysis of insect cells, so as
to provide a product which is at least substantially free of host
debris, e.g. proteins, lipids and polysaccharides.
[0144] In order to obtain protein expression, recombinant host
cells derived from the transformants are incubated under conditions
which allow expression of the recombinant protein encoding
sequence. These conditions will vary, dependent upon the host cell
selected. However, the conditions are readily ascertainable to
those of ordinary skill in the art, based upon what is known in the
art.
[0145] iii. Plant Systems
[0146] There are many plant cell culture and whole plant genetic
expression systems known in the art. Exemplary plant cellular
genetic expression systems include those described in patents, such
as: U.S. Pat. No. 5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat.
No. 5,608,143. Additional examples of genetic expression in plant
cell culture has been described by Zenk, Phylochemistry
30:3861-3863 (1991). Descriptions of plant protein signal peptides
may be found in addition to the references described above in
Vaulcombe et al., Mol. Gen. Genel. 209:33-40 (1987); Chandler et
al., Plant Molecular Biology 3:407-418 (1984); Rogers, J Biol.
Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356
(1987); Whittier et al., Nucleic Acids Research 15:2515-2535
(1987); Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et
al., Gene 122:247-253 (1992). A description of the regulation of
plant gene expression by the phytohormone, gibberellic acid and
secreted enzymes induced by gibberellic acid can be found in R. L.
Jones and J. MacMillin, Gibberellins: in: Advanced Plant
Physiology,. Malcolm B. Wilkins, ed., 1984 Pitman Publishing
Limited, London, pp. 21-52. References that describe other
metabolically-regulated genes: Sheen, Plant Cell,
2:1027-1038(1990); Maas et al., EMBO J. 9:3447-3452 (1990); Benkel
& Hickey, PNAS USA 84:1337-1339 (1987) Typically, using
techniques known in the art, a desired polynucleotide sequence is
inserted into an expression cassette comprising genetic regulatory
elements designed for operation in plants. The expression cassette
is inserted into a desired expression vector with companion
sequences upstream and downstream from the expression cassette
suitable for expression in a plant host. The companion sequences
will be of plasmid or viral origin and provide necessary
characteristics to the vector to permit the vectors to move DNA
from an original cloning host, such as bacteria, to the desired
plant host. The basic bacterial/plant vector construct will
preferably provide a broad host range prokaryote replication
origin; a prokaryote selectable marker, and, for Agrobacterium
transformations, T DNA sequences for Agrobacterium-mediated
transfer to plant chromosomes. Where the heterologous gene is not
readily amenable to detection, the construct will preferably also
have a selectable marker gene suitable for determining if a plant
cell has been transformed. A general review of suitable markers,
e.g. for the members of the grass family, is found in Wilmink &
Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.
[0147] Sequences suitable for permitting integration of the
heterologous sequence into the plant genome are also recommended.
These might include transposon sequences and the like for
homologous recombination as well as Ti sequences which permit
random insertion of a heterologous expression cassette into a plant
genome. Suitable prokaryote selectable markers include resistance
toward antibiotics such as ampicillin or tetracycline. Other DNA
sequences encoding additional functions may also be present in the
vector, as is known in the art.
[0148] The nucleic acid molecules of the subject invention may be
included into an expression cassette for expression of the
protein(s) of interest. Usually, there will be only one expression
cassette, although two or more are feasible. The recombinant
expression cassette will contain in addition to the heterologous
protein encoding sequence the following elements, a promoter
region, plant 5' untranslated sequences, initiation codon depending
upon whether or not the structural gene comes equipped with one,
and a transcription and translation termination sequence. Unique
restriction enzyme sites at the 5' and 3' ends of the cassette
allow for easy insertion into a pre-existing vector.
[0149] A heterologous coding sequence may be for any protein
relating to the present invention. The sequence encoding the
protein of interest will encode a signal peptide which allows
processing and translocation of the protein, as appropriate, and
will usually lack any sequence which might result in the binding of
the desired protein of the invention to a membrane. Since, for the
most part, the transcriptional initiation region will be for a gene
which is expressed and translocated during germination, by
employing the signal peptide which provides for translocation, one
may also provide for translocation of the protein of interest. In
this way, the protein(s) of interest will be translocated from the
cells in which they are expressed and may be efficiently harvested.
Typically secretion in seeds are across the aleurone or scutellar
epithelium layer into the endosperm of the seed. While it is not
required that the protein be secreted from the cells in which the
protein is produced, this facilitates the isolation and
purification of the recombinant protein.
[0150] Since the ultimate expression of the desired gene product
will be in a eucaryotic cell it is desirable to determine whether
any portion of the cloned gene contains sequences which will be
processed out as introns by the host's splicosome machinery. If so,
site-directed mutagenesis of the "intron" region may be conducted
to prevent losing a portion of the genetic message as a false
intron code, Reed and Maniatis, Cell 41:95-105, 1985.
[0151] The vector can be microinjected directly into plant cells by
use of micropipettes to mechanically transfer the recombinant DNA.
Crossway, Mol. Gen Genet, 202:179-185, 1985. The genetic material
may also be transferred into the plant cell by using polyethylene
glycol, Krens, et al., Nature, 296, 72-74, 1982. Another method of
introduction of nucleic acid segments is high velocity ballistic
penetration by small particles with the nucleic acid either within
the matrix of small beads or particles, or on the surface, Klein,
et al., Nature, 327, 70-73, 1987 and Knudsen and Muller, 1991,
Planta, 185:330-336 teaching particle bombardment of barley
endosperm to create transgenic barley. Yet another method of
introduction would be fusion of protoplasts with other entities,
either minicells, cells, lysosomes or other fusible lipid-surfaced
bodies, Fraley, et al., PNAS USA, 79, 1859-1863, 1982.
[0152] The vector may also be introduced into the plant cells by
electroporation. (Fromm et al., PNAS USA 82:5824, 1985). In this
technique, plant protoplasts are electroporated in the presence of
plasmids containing the gene construct. Electrical impulses of high
field strength reversibly permeabilize biomembranes allowing the
introduction of the plasmids. Electroporated plant protoplasts
reform the cell wall, divide, and form plant callus.
[0153] All plants from which protoplasts can be isolated and
cultured to give whole regenerated plants can be transformed by the
present invention so that whole plants are recovered which contain
the transferred gene. It is known that practically all plants can
be regenerated from cultured cells or tissues, including but not
limited to all major species of sugarcane, sugar beet, cotton,
fruit and other trees, legumes and vegetables. Some suitable plants
include, for example, species from the genera Fragaria, Lotus,
Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum,
Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus,
Sinapis, Atropa, Capsicum, Dalura, Hyoscyamus, Lycopersion,
Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium,
Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis,
Nemesia, Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio,
Solpiglossis, Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum,
Sorghum, and Datura.
[0154] Means for regeneration vary from species to species of
plants, but generally a suspension of transformed protoplasts
containing copies of the heterologous gene is first provided.
Callus tissue is formed and shoots may be induced from callus and
subsequently rooted. Alternatively, embryo formation can be induced
from the protoplast suspension. These embryos germinate as natural
embryos to form plants. The culture media will generally contain
various amino acids and hormones, such as auxin and cytokinins. It
is also advantageous to add glutamic acid and proline to the
medium, especially for such species as corn and alfalfa. Shoots and
roots normally develop simultaneously. Efficient regeneration will
depend on the medium, on the genotype, and on the history of the
culture. If these three variables are controlled, then regeneration
is fully reproducible and repeatable.
[0155] In some plant cell culture systems, the desired protein of
the invention may be excreted or alternatively, the protein may be
extracted from the whole plant. Where the desired protein of the
invention is secreted into the medium, it may be collected.
Alternatively, the embryos and embryoless-half seeds or other plant
tissue may be mechanically disrupted to release any secreted
protein between cells and tissues. The mixture may be suspended in
a buffer solution to retrieve soluble proteins. Conventional
protein isolation and purification methods will be then used to
purify the recombinant protein. Parameters of time, temperature pH,
oxygen, and volumes will be adjusted through routine methods to
optimize expression and recovery of heterologous protein.
[0156] iv. Bacterial Systems
[0157] Bacterial expression techniques are known in the art. A
bacterial promoter is any DNA sequence capable of binding bacterial
RNA polymerase and initiating the downstream (3') transcription of
a coding sequence (e.g. structural gene) into mRNA. A promoter will
have a transcription initiation region which is usually placed
proximal to the 5'end of the coding sequence. This transcription
initiation region usually includes an RNA polymerase binding site
and a transcription initiation site. A bacterial promoter may also
have a second domain called an operator, that may overlap an
adjacent RNA polymerase binding site at which RNA synthesis begins.
The operator permits negative regulated (inducible) transcription,
as a gene repressor protein may bind the operator and thereby
inhibit transcription of a specific gene. Constitutive expression
may occur in the absence of negative regulatory elements, such as
the operator. In addition, positive regulation may be achieved by a
gene activator protein binding sequence, which, if present is
usually proximal (5') to the RNA polymerase binding sequence. An
example of a gene activator protein is the catabolite activator
protein (CAP), which helps initiate transcription of the lac operon
in E.coli [Raibaud et al. (1984) Annu. Rev. Genel. 18:173].
Regulated expression may therefore be either positive or negative,
thereby either enhancing or reducing transcription.
[0158] Sequences encoding metabolic pathway enzymes provide
particularly useful promoter sequences. Examples include promoter
sequences derived from sugar metabolizing enzymes, such as
galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and
maltose. Additional examples include promoter sequences derived
from biosynthetic enzymes such as tryptophan (trp) [Goeddel et al.
(1980) Nuc. Acids Res. 8:4057; Yelverton et al. (1981) Nucl Acids
Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 and
EP-A-0121775]. The g-lactamase (bla) promoter system [Weissmann
(1981) "The cloning of interferon and other mistakes." In
Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake
et al. (1981) Nature 292:128] and T5 [U.S. Pat. No. 4,689,406]
promoter systems also provide useful promoter sequences.
[0159] In addition, synthetic promoters which do not occur in
nature also function as bacterial promoters. For example,
transcription activation sequences of a bacterial or bacteriophage
promoter may be joined with the operon sequences of another
bacterial or bacteriophage promoter, creating a synthetic hybrid
promoter [U.S. Pat. No. 4,551,433]. For example, the tac promoter
is a hybrid trp-lac promoter comprised of both trp promoter and lac
operon sequences that is regulated by the lac repressor [Amann et
al. (1983) Gene 25:167; de Boer el al. (1983) PNAS USA 80:21].
Furthermore, a bacterial promoter can include naturally occurring
promoters of non-bacterial origin that have the ability to bind
bacterial RNA polymerase and initiate transcription. A naturally
occurring promoter of non-bacterial origin can also be coupled with
a compatible RNA polymerase to produce high levels of expression of
some genes in prokaryotes. The bacteriophage T7 RNA
polymerase/promoter system is an example of a coupled promoter
system [Studier et al. (1986) J.Mol.Biol. 189:113; Tabor et al.
(1985) PNAS USA 82:1074]. In addition, a hybrid promoter can also
be comprised of a bacteriophage promoter and an E.coli operator
region (EP-A-0267851).
[0160] In addition to a functioning promoter sequence, an efficient
ribosome binding site is also useful for the expression of foreign
genes in prokaryotes. In E.coli the ribosome binding site is called
the Shine-Dalgamo (SD) sequence and includes an initiation codon
(ATG) and a sequence 3-9 nucleotides in length located 3-11
nucleotides upstream of the initiation codon [Shine et al. (1975)
Nature 254:34]. The SD sequence is thought to promote binding of
mRNA to the ribosome by base-pairing between the SD sequence and
the 3' end of 16S rRNA [Steitz et al. (1979) "Genetic signals and
nucleotide sequences in messenger RNA." In Biological Regulation
and Development: Gene Expression (ed. R.F. Goldberger)]. To express
eukaryotic genes and prokaryotic genes with weak ribosome-binding
site [Sambrook et a. (1989) "Expression of cloned genes in
Escherichia coli." In Molecular Cloning: A Laboratory Manual].
[0161] A DNA molecule may be expressed intracellularly. A promoter
sequence may be directly linked with the DNA molecule, in which
case the first amino acid at the N-terminus will always be a
methionine, which is encoded by the ATG start codon. If desired,
methionine at the N-terminus may be cleaved from the protein by in
vitro incubation with cyanogen bromide or by either in vivo on in
vitro incubation with a bacterial methionine N-terminal peptidase
(EPO-A-0 219 237).
[0162] Fusion proteins provide an alternative to direct expression.
Usually, a DNA sequence encoding the N-terminal portion of an
endogenous bacterial protein, or other stable protein, is fused to
the 5' end of heterologous coding sequences. Upon expression, this
construct will provide a fusion of the two amino acid sequences.
For example, the bacteriophage lambda cell gene can be linked at
the 5'terminus of a foreign gene and expressed in bacteria. The
resulting fusion protein preferably retains a site for a processing
enzyme (factor Xa) to cleave the bacteriophage protein from the
foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins
can also be made with sequences from the lacZ [Jia et al. (1987)
Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93;
Makoffet et al. (1989) J. Gen Microbiol. 135:11], and Chey [EP-A-0
324 647] genes. The DNA sequence at the junction of the two amino
acid sequences may or may not encode a cleavable site. Another
example is a ubiquitin fusion protein. Such a fusion protein is
made with the ubiquitin region that preferably retains a site for a
processing enzyme (e.g. ubiquitin specific processing-protease) to
cleave the ubiquitin from the foreign protein. Through this method,
native foreign protein can be isolated [Miller et al. (1989)
Bio/Technology 7:698].
[0163] Alternatively, foreign proteins can also be secreted from
the cell by creating chimeric DNA molecules that encode a fusion
protein comprised of a signal peptide sequence fragment that
provides for secretion of the foreign protein in bacteria [U.S.
Pat. No. 4,336,336]. The signal sequence fragment usually encodes a
signal peptide comprised of hydrophobic amino acids which direct
the secretion of the protein from the cell. The protein is either
secreted into the growth media (gram-positive bacteria) or into the
periplasmic space, located between the inner and outer membrane of
the cell (gram-negative bacteria). Preferably there are processing
sites, which can be cleaved either in vivo or in vitro encoded
between the signal peptide fragment and the foreign gene.
[0164] DNA encoding suitable signal sequences can be derived from
genes for secreted bacterial proteins, such as E.coli outer
membrane protein gene (ompA) [Masui et al. (1983) in: Experimental
Manipulation of Gene Expression; Ghrayeb et al. (1984) EMBO J
3:2437] and the E.coli alkaline phosphatase signal sequence (phoA)
[Oka et al. (1985) PNAS USA 82:7212]. As a further example, signal
sequence of the alpha-amylase gene from various Bacillus strains
can be used to secrete heterologous proteins from B.subtilis [Palva
et al. (1982) PNAS USA 79:5582; EP-A-02440421].
[0165] Usually, transcription termination sequences recognized by
bacteria are regulatory regions located 3' to the translation stop
codon, and thus together with the promoter flank the coding
sequence. These sequences direct the transcription of an mRNA which
can be translated into the polypeptide encoded by the DNA.
Transcription termination sequences frequently include DNA
sequences of about 50 nucleotides capable of forming stem loop
structures that aid in terminating transcription. Examples include
transcription termination sequences derived from genes with strong
promoters, such as the trp gene in E.coli as well as other
biosynthetic genes.
[0166] Usually, the above described components, comprising a
promoter, signal sequence (if desired), coding sequence of
interest, and transcription termination sequence, are put together
into expression constructs. Expression constructs are often
maintained in a replicon, such as an extrachromosomal element (e.g.
plasmids) capable of stable maintenance in a host, such as
bacteria. The replicon will have a replication system, thus
allowing it to be maintained in a prokaryotic host either for
expression or for cloning and amplification. In addition, a
replicon may be either a high or low copy number plasmid. A high
copy number plasmid will generally have a copy number ranging from
.about.5 to .about.200, and usually .about.10 to .about.150. A host
containing a high copy number plasmid will preferably contain at
least .about.10, and more preferably at least .about.20 plasmids.
Either a high or low copy number vector may be selected, depending
upon the effect of the vector and the foreign protein on the
host.
[0167] Alternatively, the expression constructs can be integrated
into the bacterial genome with an integrating vector. Integrating
vectors usually contain at least one sequence homologous to the
bacterial chromosome that allows the vector to integrate.
Integrations appear to result from recombinations between
homologous DNA in the vector and the bacterial chromosome. For
example, integrating vectors constructed with DNA from various
Bacillus strains integrate into the Bacillus chromosome (EP-A-01
27328). Integrating vectors may also be comprised of bacteriophage
or transposon sequences.
[0168] Usually, extrachromosomal and integrating expression
constructs may contain selectable markers to allow for the
selection of bacterial strains that have been transformed.
Selectable markers can be expressed in the bacterial host and may
include genes which render bacteria resistant to drugs such as
ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin),
and tetracycline [Davies et al. (1978) Annu. Rev. Microbiol.
32:469]. Selectable markers may also include biosynthetic genes,
such as those in the histidine, tryptophan, and leucine
biosynthetic pathways.
[0169] Alternatively, some of the above described components can be
put together in transformation vectors. Transformation vectors are
usually comprised of a selectable market that is either maintained
in a replicon or developed into an integrating vector, as described
above.
[0170] Expression and transformation vectors, either
extra-chromosomal replicons or integrating vectors, have been
developed for transformation into many bacteria. For example,
expression vectors have been developed for, inter alia, the
following bacteria: Bacillus subtilis [Palva et. al. (1982) PNAS
USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO 84/04541],
Escherichia coli [Shimatake et al. (1981) Nature 292:128; Amann el
al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol.
189:113; EP-A-0 036 776,EP-A-0 136 829 and EP-A-0 136 907],
Streptococcus cremoris [Powell et al. (1988) Appl. Environ.
Microbial. 54:655]; Streptococcus lividans [Powell el al. (1988)
Appl. Environ Microbial. 54:655], Streptomyces lividans [U.S. Pat.
No. 4,745,056].
[0171] Methods of introducing exogenous DNA into bacterial hosts
are well-known in the art, and usually include either the
transformation of bacteria treated with CaCl.sub.2 or other agents,
such as divalent cations and DMSO. DNA can also be introduced into
bacterial cells by electroporation. Transformation procedures
usually vary with the bacterial species to be transformed. See e.g.
[Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al.
(1982) PNAS USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO
84/04541, Bacillus], [Miller et al. (1988) PNAS USA 85:856; Wang el
al. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al.
(1973) PNAS USA 69:2110; Dower et al. (1988) Nucleic Acids Res.
16:6127; Kushner (1978) "An improved method for transformation of
Escherichia coli with ColE1-derived plasmids. In Genetic
Engineering: Proceedings of the International Symposium on Genetic
Engineering (eds. H.W. Boyer and S. Nicosia); Mandel et al.
(1970)J. Mol. Biol. 53:159; Taketo (1988) Biochim. Biophys. Acta
949:318; Escherichia], [Chassy el al. (1987) FEMS MicrobioL Lett.
44:173 Lactobacillus]; [Fiedler et. al. (1988) AnaL Biochem 170:38,
Pseudomonas]; [Augustin et al. (1990) FEMS MicrobioL Lett. 66:203,
Staphylococcus], [Barany et al. (1980) J. Bacteriol. 144:698;
Hariander (1987) "Transformation of Streptococcus lactis by
electroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.
Curtiss III); Perry et al. (1981) Infect. Immun 32:1295; Powellet
et al. (1988) Appl. Environ. Microbiol. 54:655; Somkutiet al.
(1987) Proc. 4th Evr. Cong. Biotechnology 1:412,
Streptococcus].
[0172] General guidance on expression in E.coli and its
optimisation can be found in Baneyx (1999) Curr.Opin.Biolech.
10:411421 and Hannig & Makrides (1998) TIBTECH 16:54-60.
[0173] v. Yeast Expression
[0174] Yeast expression systems are also known to one of ordinary
skill in the art. A yeast promoter is any DNA sequence capable of
binding yeast RNA polymerase and initiating the downstream (3')
transcription of a coding sequence (e.g. structural gene) into
mRNA. A promoter will have a transcription initiation region which
is usually placed proximal to the 5' end of the coding sequence.
This transcription initiation region usually includes an RNA
polymerase binding site (the "TATA Box") and a transcription
initiation site. A yeast promoter may also have a second domain
called an upstream activator sequence (UAS), which, if present, is
usually distal to the structural gene. The UAS permits regulated
(inducible) expression. Constitutive expression occurs in the
absence of a UAS. Regulated expression may be either positive or
negative, thereby either enhancing or reducing transcription.
[0175] Yeast is a fermenting organism with an active metabolic
pathway, therefore sequences encoding enzymes in the metabolic
pathway provide particularly useful promoter sequences. Examples
include alcohol dehydrogenase (ADH) (EP-A-0284044),
glucose-6-phosphate isomerase,
glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH),
hexokinase, phosphofructokinase, 3-phosphoglycerate mutase,
enolase, glucokinase, and pyruvate kinase (PyK) (EPO-A-0329203).
The yeast PHO5 gene, encoding acid phosphatase, also provides
useful promoter sequences [Myanohara et al. (1983) PNAS USA
80:1].
[0176] In addition, synthetic promoters which do not occur in
nature also function as yeast promoters. For example, UAS sequences
of one yeast promoter may be joined with the transcription
activation region of another yeast promoter, creating a synthetic
hybrid promoter. Examples of such hybrid promoters include the ADH
regulatory sequence linked to the GAP transcription activation
region (U.S. Pat. Nos. 4,876,197 and 4,880,734). Other examples of
hybrid promoters include promoters which consist of the regulatory
sequences of either the ADH2, GAL4, GAL10, OR PHO5 genes, combined
with the transcriptional activation region of a glycolytic enzyme
gene such as GAP or PyK (EP-A-0 164 556). Furthermore, a yeast
promoter can include naturally occurring promoters of non-yeast
origin that have the ability to bind yeast RNA polymerase and
initiate transcription. Examples of such promoters include, inter
alia, [Cohen et al. (1980)PNAS USA 77:1078; Henikoff et al. (1981)
Nature 283:835; Hollenberg et al. (1981) Curr. Topics MicrobioL
Immunol. 96:119; Hollenberg et al. (1979) "The Expression of
Bacterial Antibiotic Resistance Genes in the Yeast Saccharomyces
cerevisiae," in: Plasmids of Medical, Environmental and Commercial
Importance (eds. K.N. Timmis and A. Puhler); Mercerau-Puigalon et
al. (1980) Gene 11:163; Panthier et al. (1980) Curr. Genel
2:109;].
[0177] A DNA molecule may be expressed intracellularly in yeast. A
promoter sequence may be directly linked with the DNA molecule, in
which case the first amino acid at the N-terminus of the
recombinant protein will always be a methionine, which is encoded
by the ATG start codon. If desired, methionine at the N-terminus
may be cleaved from the protein by in vitro incubation with
cyanogen bromide.
[0178] Fusion proteins provide an alternative for yeast expression
systems, as well as in mammalian, baculovirus, and bacterial
expression systems. Usually, a DNA sequence encoding the N-terminal
portion of an endogenous yeast protein, or other stable protein, is
fused to the 5' end of heterologous coding sequences. Upon
expression, this construct will provide a fusion of the two amino
acid sequences. For example, the yeast or human superoxide
dismutase (SOD) gene, can be linked at the 5' terminus of a foreign
gene and expressed in yeast. The DNA sequence at the junction of
the two amino acid sequences may or may not encode a cleavable
site. See e.g. EP-A-0 196 056. Another example is a ubiquitin
fusion protein. Such a fusion protein is made with the ubiquitin
region that preferably retains a site for a processing enzyme (e.g.
ubiquitin-specific processing protease) to cleave the ubiquitin
from the foreign protein. Through this method, therefore, native
foreign protein can be isolated (e.g. WO88/024066).
[0179] Alternatively, foreign proteins can also be secreted from
the cell into the growth media by creating chimeric DNA molecules
that encode a fusion protein comprised of a leader sequence
fragment that provide for secretion in yeast of the foreign
protein. Preferably, there are processing sites encoded between the
leader fragment and the foreign gene that can be cleaved either in
vivo or in vitro. The leader sequence fragment usually encodes a
signal peptide comprised of hydrophobic amino acids which direct
the secretion of the protein from the cell.
[0180] DNA encoding suitable signal sequences can be derived from
genes for secreted yeast proteins, such as the invertase gene
(EP-A-0 012 873; JPO. 62,096,086) and the A-factor gene (U.S. Pat.
No. 4,588,684). Alternatively, leaders of non-yeast origin, such as
an interferon leader, exist that also provide for secretion in
yeast (EP-A-0 060 057).
[0181] A preferred class of secretion leaders are those that employ
a fragment of the yeast alpha-factor gene, which contains both a
"pre" signal sequence, and a "pro" region. The types of
alpha-factor fragments that can be employed include the full-length
pre-pro alpha factor leader (about 83 aa residues) as well as
truncated alpha-factor leaders (usually about 25 to about 50 amino
acid residues) (U.S. Pat. No. 4,546,083 and 4,870,008; EP-A-0 324
274). Additional leaders employing an alpha-factor leader fragment
that provides for secretion include hybrid alpha-factor leaders
made with a presequence of a first yeast, but a pro-region from a
second yeast alphafactor. (e.g. see WO 89/02463.)
[0182] Usually, transcription termination sequences recognized by
yeast are regulatory regions located 3'to the translation stop
codon, and thus together with the promoter flank the coding
sequence. These sequences direct the transcription of an mRNA which
can be translated into the polypeptide encoded by the DNA. Examples
of transcription terminator sequence and other yeast-recognized
termination sequences, such as those coding for glycolytic
enzymes.
[0183] Usually, these components, comprising a promoter, leader (if
desired), coding sequence of interest, and transcription
termination sequence, are put together into expression constructs.
Expression constructs are often maintained in a replicon, such as
an extrachromosomal element (e.g. plasmids) capable of stable
maintenance in a host, such as yeast or bacteria. The replicon may
have two replication systems, thus allowing it to be maintained,
for example, in yeast for expression and in a prokaryotic host for
cloning and amplification. Examples of such yeast-bacteria shuttle
vectors include YEp24 [Botsteinet et al. (1979) Gene 8:17-24],
pCl/l [Brake et al. (1984) PNAS USA 81:4642-4646], and YRp17
[Stinchcomb et al. (1982) J. Mol. Biol. 158:157). In addition, a
replicon may be either a high or low copy number plasmid. A high
copy number plasmid will generally have a copy number ranging from
.about.5 to .about.200, and usually .about.10 to .about.150. A host
containing a high copy number plasmid will preferably have at least
.about.10, and more preferably at least .about.20. Either a high or
low copy number vector may be selected, depending upon the effect
of the vector and the foreign protein on the host. See e.g. Brakeet
et al., supra.
[0184] Alternatively, the expression constructs can be integrated
into the yeast genome with an integrating vector. Integrating
vectors usually contain at least one sequence homologous to a yeast
chromosome that allows the vector to integrate, and preferably
contain two homologous sequences flanking the expression construct.
Integrations appear to result from recombinations between
homologous DNA in the vector and the yeast chromosome [Orr-Weaver
et al. (1983) Methods in Enzymol. 101:228-245]. An integrating
vector may be directed to a specific locus in yeast by selecting
the appropriate homologous sequence for inclusion in the vector.
See Orr-Weaver et al., supra. One or more expression construct may
integrate, possibly affecting levels of recombinant protein
produced [Rine et al. (1983) PNAS USA 80:6750]. The chromosomal
sequences included in the vector can occur either as a single
segment in the vector, which results in the integration of the
entire vector, or two segments homologous to adjacent segments in
the chromosome and flanking the expression construct in the vector,
which can result in the stable integration of only the expression
construct.
[0185] Usually, extrachromosomal and integrating expression
constructs may contain selectable markers to allow for the
selection of yeast strains that have been transformed. Selectable
markers may include biosynthetic genes that can be expressed in the
yeast host, such as ADE2, HIS4, LEU2, TRP1, and ALG7, and the G418
resistance gene, which confer resistance in yeast cells to
tunicamycin and G418, respectively. In addition, a suitable
selectable marker may also provide yeast with the ability to grow
in the presence of toxic compounds, such as metal. For example, the
presence of CUP1 allows yeast to grow in the presence of copper
ions [Butt et al. (1987) Microbiol, Rev. 51:351].
[0186] Alternatively, some of the above described components can be
put together into transformation vectors. Such vectors usually
comprise a selectable marker that is either maintained in a
replicon or developed into an integrating vector as described
above. Expression and transformation vectors, either
extrachromosomal replicons or integrating vectors, have been
developed for transfomnation into many yeasts. For example,
expression vectors have been developed for, inter alia, the
following yeasts:Candida albicans [Kurtz, et al. (1986) Mol. Cell.
Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. Basic
Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J.
Gen Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet.
202:302], Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol.
158:1165], Kluyveromyces lactis [De Louvencourt et al. (1983) J.
Bacteriol. 154:737; Van den Berg et al. (1990) Bio/Technology
8:135], Pichia guillerimondii [Kunze et al. (1985) J. Basic
Microbiol. 25:141], Pichia pastoris [Cregg, et al. (1985) Mol.
Cell. Biol. 5:3376; U.S. Pat. Nos. 4,837,148 and 4,929,555,],
Saccharomyces cerevisiae [Hinnen et al. (1978) PNAS USA 75:1929;
Ito et al. (1983) J. Bacteriol. 153:163], Schizosaccharomyces pombe
[Beach and Nurse (1981) Nature 300:706], and Yarrowia lipolytica
[Davidow, et al. (1985) Curr. Genet. 10:380471 Gaillardin, et al.
(1985) Curr. Genet. 10:49].
[0187] Methods of introducing exogenous DNA into yeast hosts are
well-known in the art, and usually include either the
transformation of spheroplasts or of intact yeast cells treated
with alkali cations. Transformation procedures usually vary with
the yeast species to be transformed. See e.g. [Kurtz et al. (1986)
Mol. Cell. Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol.
25:141; Candida]; [Gleeson et al. (1986) J. Gen Microbiol.
132:3459; Roggenkamp et al. (1986) Mol. Gen Genet. 202:302;
Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De
Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et
al. (1990) Bio/Technology 8:135; Kluyveromyces]; [Cregg et al.
(1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic
Microbiol. 25:141; U.S. Pat. Nos. 4,837,148 and 4,929,555; Pichia];
[Hinnen et al. (1978) PNAS USA 75;1929; Ito et al. (1983)J.
Bacteriol. 153:163 Saccharomyces]; [Beach and Nurse (1981) Nature
300:706; Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet.
10:39; Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].
BRIEF DESCRIPTION OF DRAWINGS
[0188] FIG. 1 shows structural and functional relationships of
Pichia anomala killer toxin (PaKT), PaKT-neutralizing monoclonal
antibody (mAb KT4), killer toxin receptor (KTR), and PaKT-like
killer antibodies and derivative killer mimotopes (KM).
[0189] FIG. 2 shows in vitro microbicidal activity by CFU assay of
mAb K10 and mAb K20 in comparison with an irrelevant
isotype-matched mAb (all at 100 .mu.g/ml dose) against Candida
albicans UP10S.
[0190] FIG. 3 shows in vitro microbicidal activity of scFv
antibodies H6 and H20 (100 .mu.g/ml dose).
[0191] FIG. 4 shows in vitro microbicidal activity of peptides KM2
and KM3 compared to KM0 and IP controls (1 mg/ml dose).
[0192] FIG. 5 shows in vitro microbicidal activity of KM4 (0.5
mg/ml dose).
[0193] FIG. 6 shows in vitro microbicidal activity of KM5 (1 mg/ml
dose).
[0194] FIG. 7 shows in vitro microbicidal activity of KM1 (1 mg/ml
dose).
[0195] FIG. 8 shows clearance of vaginal candidiasis in rats
intravaginally administered with KM.
[0196] FIG. 9 shows a Kaplan-Meyer survival curve of SCID mice
challenged with 5 LD50 of C. albicans and treated with KM.
[0197] FIG. 10 shows microbicidal activity of KM (25 & 10
.mu.g/ml doses) against C.neoformans UP25.
[0198] FIG. 11 shows microbicidal activity of KM (500 .mu.g/ml
& 100 .mu.g/ml doses) against S.aureus a38.
[0199] FIG. 12 shows in vitro microbicidal activity of KM0, KM6, KM
and KM7 at 20 .mu.g/ml dose.
[0200] FIG. 13 is an illustration of features within the H6 scFv
(SEQ ID 2).
[0201] FIG. 14 shows the effect of KM peptide (14A & 14B) on
A.castellani growth, compared to the effect of SP peptide (14C
& 14D). Growth was at either 37.degree. C. (14A & 14C) or
25.degree. C. (14B & 14D). The graphs show the number of
trophozoites per well.
[0202] FIG. 15 shows the effect of KM peptide on A.castellani cell
viability. SP peptide is set as 100%.
[0203] FIG. 16 shows the effect of the K20 mAb on A.castellani
growth, and FIG. 17 shows the effect of K10 mAb. Values are the
number of trophozoites per well. Amoebal growth in medium alone is
shown as crosses. Antibodies were used at 12.5 .mu.g/ml
(.diamond-solid.), 25.mu.g/ml (.DELTA.) or 50 .mu.g/ml
(.smallcircle.).
[0204] FIG. 18 shows the effect of KM (circles) and SP (crosses)
peptides on influenza virus replication at up to 80 .mu.g/ml
concentration. Values are log.sub.2 HA titres.
[0205] FIG. 19 shows the effect of KM (circles) and SP (squares)
peptides on HIV-1 replication. Peptides were used at either 1
.mu.g/ml (closed) or 10 .mu.g/ml (open). Values are copies/ml over
15 days of culture.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0206] TABLE-US-00005 BRIEF DESCRIPTION OF THE SEQUENCE LISTING SEQ
ID Description 1 Nucleotide sequence of H6 scFv 2 Amino acid
sequence of H6 scFv 3 KM0 (fragment of SEQ ID 2) 4 KM (=SEQ ID 3
with Glu.fwdarw.Ala mutation at position 1) 5-12 Alanine-scanning
variants of SEQ ID 3 13 SP0 scramble peptide 14-20 Shortened
derivatives of KM (9mer, 8mer, 7mer, 6mer, 5mer, 4mer, 3mer from
SEQ ID 3) 21 Nucleotide sequence of H20 scFv 22 Amino acid sequence
of H20 scFv 23 KM1 (=SEQ ID 3 with Cys.fwdarw.Ser mutation at
position 7) 24 KM2 25 KM3 26 KM4 27 KM5 28 Irrelevant peptide IP 29
SP 30 Linker (used to join KM0 and KM2 to give KM3) 31 Peptide
control 32 KM1 derivative (=SEQ ID 4 with Cys.fwdarw.Xaa mutation
at position 7) 33 SEQ ID 32 with Cys.fwdarw.Ser mutation at
position 7 34-38 KM0, KM1, KM2, KM4 & KM5 equivalents from H20
39 KM equivalent from H20 40-47 Alanine-scanning variants of SEQ ID
24 48 KM2 (SEQ ID 24) with Cys.fwdarw.Ser mutation at position 7
49-57 Alanine-scanning variants of SEQ ID 27 58 KM4 (SEQ ID 26)
with Cys.fwdarw.Ser mutation at position 7 59 C-terminus `E-tag`
from scFv system 60-65 CDRs from H6 antibody (fragments of SEQ ID
2) 66-71 CDRs from H20 antibody (fragments of SEQ ID 22) NB: the
inclusion of a polypeptide sequence in the sequence listing does
not imply any particular D- or L-configuration to its constituent
amino acids.
MODES FOR CARRYING OUT THE INVENTION
[0207] H6 Single-Chain Fv Antibody
[0208] H6 is a single-chain Fv raised against the idiotope of the
KT4 monoclonal antibody. It is an anti-idiotypic antibody raised
with the purpose of mimicking the activity of the Pichia anomala
killer toxin (PaKT).
[0209] The existence of scFv H6 has previously been reported [e.g.
ref. 13], but a method for its manufacture has not previously been
disclosed, nor has its amino acid sequence. The sequence of H6 is
now disclosed (SEQ IDs 1 and 2). Within SEQ ID 2, amino acids
107-132 (GT . . . IE) are a linker and the final 13 amino acids (GA
. . . PR; SEQ ID 59) are the `E-tag` inserted by the Recombinant
Phage Antibody System (Pharmacia Biotech.TM.) used to create the
scFv.
[0210] The CDRs within H6 are as follows: TABLE-US-00006 CDR aa SEQ
ID H1 33-37 60 H2 52-65 61 H3 98-101 62 L1 153-162 63 L2 178-184 64
L3 217-224 65
[0211] The H6 scFv has strong microbicidal effects in vitro against
important pathogenic microorganisms including: C.albicans; C.krusei
and C. glabrata (including fluconazole-resistant strains);
Cryptococcus neoformans; A. fumigatus; M.tuberculosis; S.aureus,
Enterococcus faecalis, E.faecium, and Streptococcus pneumoniae
(including methicillin-, vancomycin- and penicillin-resistant
strains); S.mutans, Leishmania major, L.infantum and Achantamoeba
castellani. Furthermore, it shows specific therapeutic activity in
an in vivo model of rat vaginal candidiasis by intravaginal
administration.
[0212] K20 Monoclonal Antibody and scFv Derivative H20
[0213] K10 is an anti-idiotypic rat monoclonal antibody raised
against KT4. Like the H6 scFv, it shows good in vitro microbicidal
activity. In addition, it has been shown to be therapeutic against
P.carinii pneumonia in rats infected by aerosol administration
[16], and in mice transplanted with T cell depleted bone marrow
against aspergillosis caused by nasal instillation [14].
[0214] Anti-idiotypic antibodies were raised in mice against K10
(i.e. anti-anti-anti-idiotypic with respect to PaKT). One of the
resulting hybridoma-produced antibodies was designated `K20`.
[0215] K20 was tested in a conventional in vitro colony forming
unit (CFU) assay to evaluate killer activity. Approximately 250
viable PaKT-susceptible germinating C.albicans UP10 cells,
suspended in 10 .mu.l PBS, were added to 90 .mu.l K20 to obtain a
final concentration of 100 .mu.g/ml and incubated for 6 h at
37.degree. C. After incubation with the respective reagents, the
fungal cells were dispensed and streaked on the surface of
Sabouraud dextrose agar plates, which were therefore incubated at
30.degree. C. and their colony forming units (CFU) enumerated after
48 hours. Each experiment was performed in triplicate. An
irrelevant mAb was used as a control.
[0216] As shown in FIG. 2, K20 shows slightly better anti-candida
activity than K10.
[0217] K20 was converted into a scFv antibody using the Recombinant
Phage Antibody System (Pharmacia Biotech.TM.). The scFv was
designated `H20` and its sequence is given as SEQ IDs 21 and 22.
The H20 CDRs are as follows: TABLE-US-00007 CDR aa SEQ ID H1 33-37
66 H2 52-66 67 H3 99-115 68 L1 167-176 69 L2 192-198 70 L3 231-238
71
[0218] H20 was tested in the CFU assay (FIG. 3) and shows a
candidacidal activity comparable to H6.
[0219] Active Fragments of H6
[0220] Short peptide fragments of H6 were synthesised, with
emphasis on the CDR sequences.
[0221] Solid phase synthesis was carried out with a MultiSynTech
Syro automatic peptide synthesizer (Witten, Germany) employing Fmoc
chemistry with HOBt activation and Rink amide MBHA resin as solid
support. Peptides were cleaved from the resins and deprotected by
treatment with trifluoroacetic acid containing ethandithiol, water,
triisobutylsilane and anisole (93/2.5/2/1.5/1). After precipitation
by ethylic ether, peptides were purified by a Vydac C18 column (25
cm x lcm) and characterized by amino acid analysis and mass
spectrometry. The following peptides were synthesised:
TABLE-US-00008 # Name SEQ ID H6 residues Based on CDR 1 KM0 3
146-155 L1 2 KM2 24 91-100 H3 3 KM4 26 146-160 L1 4 KM5 27 155-163
L1
[0222] These peptides were tested in the CFU assay to evaluate
killer activity.
[0223] As a control, a `scramble` peptide (SEQ ID 13; SP0`) was
synthesised in which the amino acids of KM0 were re-ordered to have
the same overall peptide composition but different sequence. This
was used in the above assay at the same concentration as the test
KM peptides as a control for KM0 and KM4. For KM2, the control was
SEQ ID 28 (`IP` or `irrelevant peptide` ). For KM5, the control was
SEQ ID 31.
[0224] Results with KM0 were as follows, expressed as % growth in
comparison to the control: TABLE-US-00009 Peptide 100 .mu.g/ml 25
.mu.g/ml 6.25 .mu.g/ml SP0 Control 100 100 100 KM0 5.69 .+-. 0.20
29.84 .+-. 10.63 67.13 .+-. 13.81
[0225] Results with KM0, KM2, KM4 and KM5 at various concentrations
are shown in FIGS. 4 to 6. KM0 & KM5 are thus extremely
effective anti-candidal peptides. KM2 is also effective, and KM4 is
moderately effective.
[0226] The CDRs of H6 were also prepared in isolated form (SEQ IDs
60-65) and tested for microbicidal activity in the C.albicans CFU
assay. Each CDR peptide was tested at scalar dilutions to establish
the IC.sub.50. Assays were performed in triplicate and the
IC.sub.50of each peptide was calculated by nonlinear regression
analysis of curves obtained by plotting the number of CFU versus
Log peptide concentration. Results were as follows: TABLE-US-00010
Peptide IC.sub.50 (mol/l) SEQ ID Heavy chain CDR-1 2.67 .times.
10.sup.-7 60 CDR-2 1.17 .times. 10.sup.-7 61 CDR-3 1.09 .times.
10.sup.-7 62 Light chain CDR-1 6.92 .times. 10.sup.-7 63 CDR-2 2.34
.times. 10.sup.-7 64 CDR-3 2.00 .times. 10.sup.-7 65
[0227] The CDRs of H6 antibody therefore show significant
anti-candida activity, with CDR-3 from the heavy chain (SEQ ID 62)
showing the best activity.
[0228] Fragments of the H6 scFv are therefore able to act as
microbicides even though they would not be expected to hold in the
same tertiary conformation as in the intact antibody.
[0229] Polypeptides Comprising More Than One Fragment of H6
[0230] KM0 is derived from a H6 light chain CDR and KM2 is derived
from a H6 heavy chain CDR. The two decapeptides , each of which has
microbididal activity on its own, were linked by a glycine-rich
sequence (SEQ ID 30) to give KM3 (SEQ ID 25).
[0231] In comparison to IP in the CFU assay, KM3 showed
candidacidal activity, but this was weaker than either KM0 and KM2
alone (FIG. 4).
[0232] Substitution within KM0-Cysteine Replacement
[0233] The cysteine residue in decapeptide KM0 was substituted with
serine to give KM1 (SEQ ID 23), with a view to reducing oxidation
and polymerization processes. KM1 showed similar activity to KM0 in
the CFU assay (FIG. 7) in comparison to the IP control. The
substitution of cysteine with serine thus gave no apparent
alteration of the antibiotic effect of KM0, but offers increased
resistance to oxidation and thus increased in vivo half life.
[0234] SEQ ID 48 is a C.fwdarw.S substitution form of KM2. SEQ ID
58 is a C.fwdarw.S substitution form of KM4.
[0235] Substitution Within KM0-Alanine Scanning
[0236] Decapeptide KM0 was analysed by alanine scanning [32] in
order to identify the functional contributional of individual
residues to its microbicidal activity. Each of the ten constituent
amino acids was replaced with A (except for residue 9, which is
already A) and activity in the in vitro CFU assay was assessed.
Results were as follows, with values being % growth in comparison
to the SP0 control: TABLE-US-00011 SEQ ID 100 .mu.g/ml 25 .mu.g/ml
6.25 .mu.g/ml 3 5.69 .+-. 0.20 29.84 .+-. 10.63 67.13 .+-. 13.81 4
0 0 0 5 9.91 .+-. 3.28 42.67 .+-. 2.04 53.40 .+-. 6.99 6 9.27 .+-.
2.45 19.72 .+-. 2.91 60.06 .+-. 5.24 7 9.18 .+-. 4.13 26.56 .+-.
4.24 63.24 .+-. 4.77 8 0.10 .+-. 0.10 10.12 .+-. 2.96 40.42 .+-.
16.01 9 52.89 .+-. 3.90 55.52 .+-. 3.7 58.14 .+-. 8.15 10 55.71
.+-. 10.20 59.73 .+-. 4.77 64.25 .+-. 6.72 11 2.60 .+-. 0.50 23.12
.+-. 3.59 72.95 .+-. 7.42 12 11.90 .+-. 0.64 32.90 .+-. 0.79 70.33
.+-. 9.14 13 100 100 100
[0237] The most active peptide is SEQ ID 4 (`KM` ), in which the
first amino acid E is substituted by A. SEQ ID 8, in which C was
substituted by A, also shows good activity relative to both the SP0
control and to the starting KM0 decapeptide. The CFU reduction for
these two decapeptides compared to the control was statistically
significant at all three doses (p<0.005 by two-tailed Student's
t test).
[0238] On the basis of KM0 and KM sequences, scramble peptides
(SP0, SEQ ID 13; SP, SEQ ID 29) were also synthesised. Neither of
these two scramble peptides showed in vitro candidacidal
activity.
[0239] Alanine-scanning of KM2 is shown in SEQ IDs 40 to 47.
[0240] Alanine-scanning of KM5 is shown in SEQ IDs 49 to 57.
[0241] C-Terminal Truncation of KM
[0242] SEQ ID 4 (`KM`) was reduced by C-terminal deletions down to
three residues to establish the ability to retain in vitro
candidacidal activity relative to the SP control. Scalar dilutions
(100-0.8 .mu.g/ml) were tested to establish the minimal fungicidal
concentration corresponding to the killing of 100% of C. albicans
cells. KM and its trunication derivatives were also tested at
scalar dilutions to establish the peptide concentration (mol/l)
corresponding to the 50% inhibitory concentration (IC50). Assays
were performed in triplicate and the IC50 of each peptide was
calculated by nonlinear regression analysis of curves obtained by
plotting the number of CFU versus Log peptide concentration using
the GraphPad Prism 3.02 software. Results were as follows:
TABLE-US-00012 SEQ ID IC50 (mol/l) 4 5.6 .times. 10.sup.-8 14 5
.times. 10.sup.-5 15 2.3 .times. 10.sup.-5 16 6 .times. 10.sup.-7
17 7.3 .times. 10.sup.-4 18 2.5 .times. 10.sup.-5 19 7.1 .times.
10.sup.-4 20 1 .times. 10.sup.-5
[0243] There is a drop of candidacidal activity of about three
orders of magnitude with deletion of the C-terminus serine from KM.
However, the heptamer formed by deletion of the 3 C-terminal
residues shows activity only one order of magnitude lower than that
of KM.
[0244] KM Oligomers
[0245] Experiments on the stability of the killer peptide KM after
lyophilisation were carried out using the CFU assay. KM proved to
be very stable in the lyophilised form.
[0246] After solubilisation in non-reducing conditions, the free
cysteine in KM can lead to the formation of a disulfide bridge, to
give a KM dimer. The candidacidal activity of the dimer was
assessed by comparison to dimerised SP peptide. The
disulfide-bonded KM dimer retains candidacidal activity. Moreover,
this activity was maintained unaltered over a long period under
different storage conditions (4.degree. C. room temperature,
37.degree. C.).
[0247] In Vivo Activity of KM in Vaginal Infection Model
[0248] KM was tested using a well-established experimental model
[71] of vaginal C. albicans infection in oophorectomized
estrogen-treated rats. Estrogen-conditioned rats (5 animals per
group) were inoculated intravaginally with 10.sup.7 cells of
fluconazole-sensitive (SA-40) or fluconazole-resistant (AIDS 68 )
C.albicans. Both Candida strains were originally isolated from
human vaginal infection, and maintained in stock in the Department
of Bacteriology and Medical Mycology of the Instituto Superiore di
Sanita (ISS), Rome (Italy). Different doses (10, 25, 50 and 100
.mu.g) of KM were administered intravaginally at 1, 24 and 48 hours
post challenge and vaginal C.albicans burden was quantitated by CFU
enumeration from the vaginal fluid taken each day by a special
calibrated loop. Vaginal smears were also stained by PAS-van Gieson
method. Any benefit of KM treatment was assessed in terms of
microscopic reduction of the hyphal growth in the vagina. Negative
controls were untreated rats and rats treated with SP. As a
positive control, rats received 50 or 100 .mu.g/ml fluconazole
(Pfizer) in PBS (0.1 ml ) at 1, 24 and 48 hours after the yeast
challenge.
[0249] A dose-response therapeutic effect was observed at 50 and
100 .mu.g doses.
[0250] In subsequent experiments, 50 .mu.g of KM was used in the
established three dose administration schedule to determine
acceleration of fungal CFU clearance over a period of 28 days. Rats
(five per groups) were given 10.sup.7 cells in 0.1 ml of
physiological solution on day 0 and were sampled for initial
intravaginal CFU. Treatments were administered 1, 24 and 48 hours
after the challenge.
[0251] FIG. 8 shows the results of a typical experiment with strain
SA-40. At each time point there was a statistically-significant
difference (p<0.05 by two-tailed Student's t test) in vaginal
CFU counts between (a) untreated or SP-treated rats, and (b)
KM-treated or fluconazole-treated rats. There was no significant
difference between untreated and SP-treated rats, and there was no
significant difference between KM-treated and fluconazole-treated
rats.
[0252] KM significantly accelerates the early rate of clearance
(1-5 days) of the fungus from rat vagina, also providing a
substantial resolution of the infection (less than 10.sup.3 CFU/ml
of vaginal fluid) within three weeks from challenge, when the
untreated controls still had from 2 to 4.times.10.sup.4 Candida
CFU/ml of vaginal fluid. No acceleration of the fungal clearance
and no effect on resolution of infection was provided by SP
administration. The therapeutic benefit of KM was substantially
comparable with that of fluconazole.
[0253] Results for both test strains over 28 days were as follows:
TABLE-US-00013 C. albicans vaginal CFU (.times.10.sup.3) (.+-.SD)
on days: Experimental group 0 7 14 28 1. C. albicans SA-40, >100
98 .+-. 12 42 .+-. 7 38 .+-. 6 no treatment 2. C. albicans SA-40 +
>100 13 .+-. 4 2 .+-. 1 <1 50 .mu.g fluconazole 3. C.
albicans SA-40 + >100 12 .+-. 7 1 .+-. 1 <1 KM 4. C. albicans
AIDS 68, >100 70 .+-. 10 55 .+-. 8 18 .+-. 3 no treatment 5. C.
albicans AIDS >100 64 .+-. 6 48 .+-. 3 12 .+-. 4 68 + 100 .mu.g
fluconazole 6. C. albicans AIDS >100 40 .+-. 5 18 .+-. 2 <1
68 + KM
[0254] On day 7, 14 and 28, differences in CFU vaginal counts were
statistically significant (p<0.05, two-tailed Student's t test)
between: [0255] group 1 and group 2 [0256] group 1 and group 3
[0257] group 4 and group 6 [0258] group 1 and group 5 [0259] group
1 and group 6 [0260] group 5 and group 6
[0261] There was no statistically significant difference between:
[0262] group 2 and group 3 [0263] group 4 and group 5
[0264] In contrast to treatment with 100 .mu.g fluconazole, KM had
a therapeutic effect also in rat vaginal infection caused by the
fluconazole-resistant C.albicans strain.
[0265] In Vivo Anti-Candida Activity of KM in Systemic Infection
Model
[0266] KM was tested using a well-established rapidly-lethal
systemic mouse model [72] of C. albicans infection. Groups of 8
Balb/C female mice (weight 18-21 g) were challenged with 5
LD.sub.50 (10.sup.7 cells of SA-40) by the intravenous route. 50
.mu.g KM were administered intraperitoneally for three days
starting on day 0 (i.e. 1 hour after the fungal challenge) and at
24 and 48 hours thereafter. Controls were untreated animals or
animals treated with peptide SP (same dosage and treatment schedule
as those treated with KM). The animals were then followed for
mortality and internal organ invasion for 60 days. Any beneficial
effect was established in term of prolongation of the median
survival time (in days) and reduction of total mortality.
Assessment by necroscopy showed that the death of the animals was
due to the fungus, and assessment of internal organs showed
invasion by C.albicans.
[0267] In parallel experiments, SCID mice were used instead of
immunocompetent mice in order to verify whether the curative effect
of KM required the participation of host adaptive immunity. These
experiments used the same challenging fungal burden, schedule of KM
and SP treatments, and C. albicans-caused mortality end-points as
described above.
[0268] In all experiments, KM exerted a similar beneficial
therapeutic effect in terms of mortality delay and animal cure (60
days). As a typical example, FIG. 9 shows the Kaplan-Meyer survival
curve of SCID mice challenged with 5.times.LD50 of C.albicans and
treated with 50 .mu.g of either KM, fluconazole or SP, or left
untreated. KM was seen to increase the median survival time from 1
day of the untreated control to >60 days. In addition, only 1/8
KM-treated animals died as compared to 8/8 deaths in untreated mice
or those treated with SP. In all cases, death was attributable to
C.albicans challenge as shown by fungus burden in the kidneys. FIG.
9 shows that KM out-performed fluconazole.
[0269] Blocking of KM Activity by Glucans
[0270] As shown above, KM shows anti-candida activity. As KM is
distantly-related to the KT antibody, which interacts with cell
surface .beta.-glucans, the possible involvement of glucans in KM's
activity was investigated.
[0271] The binding of the KT mAb to germinating cells of C.albicans
was assessed by immunofluorescence [12]. KM completely inhibited
the binding of KT to the cells, whereas SP did not.
[0272] In further experiments, the CFU assay was performed as
before, using 25.mu.g/ml KM or SP, except that various
concentrations (between 12.5.mu.g/ml and 100 .mu.g/ml) of either
laminarin (.beta.-1,3-glucan, from Sigma) or pustulan
(.beta.-1,6-glucan, from Calbiochem) were included. Results were as
follows: TABLE-US-00014 Conc.sup.n Glucan (.mu.g/ml) CFU with KM
CFU with SP None -- 0 2822.00 .+-. 122.00 Laminarin 12.5 0 2525.33
.+-. 92.72 Laminarin 25 1.33 .+-. 0.58 2780.00 .+-. 264.21
Laminarin 50 115.33 .+-. 12.58 2742.66 .+-. 105.09 Laminarin 100
2766.66 .+-. 63.54 2740.00 .+-. 282.13 Pustulan 12.5 0 2380.00 .+-.
148.04 Pustulan 25 0 2500.00 .+-. 52.00 Pustulan 50 0 2666.00 .+-.
306.49 Pustulan 100 0 2824.00 .+-. 119.82
[0273] Thus the candidacidal activity of KM was strongly and
dose-dependently inhibited by laminarin, but not by pustulan.
[0274] These data suggest that the candidacidal KM decapeptide
competes for the binding site of KT-IdAb on fungal cell wall, and
the receptor seems to contain .beta.-1-3 glucans.
[0275] Anti-A.Castellani Activity
[0276] In addition to their anti-candida activity, the KM peptide
and the K20 monoclonal antibody were tested for activity against
trophozoites (the infectious form) of the eukaryotic free-living
soil amoeba Acanthamoeba castellanii --a cause of encephalomyelitis
and keratitis which can cause severe ocular inflammation and visual
loss.
[0277] A.castellani was grown in PYG medium at 37.degree. C. or
25.degree. C., in the presence of either SP or KM peptides at 1
.mu.g/ml, 10 .mu.g/ml or 100 .mu.g/ml (or, as a control, with no
peptide). The effect on growth over six days, in terms of the
number of trophozoites, is shown in FIG. 14. As seen in FIGS. 14C
& 14D, the scramble peptide SP has no antimicrobial activity at
either 37.degree. C. or 25.degree. C., whereas KM (FIGS. 14A &
14b) shows good antimicrobial activity.
[0278] FIG. 15 shows the in vitro activity of KM at 190 .mu.g/ml on
cell viability of A.castellanii evaluated at 25.degree. C. in
comparison with SP. FIG. 15A shows a reduction in cell viability
(p<0.05) after six hours of co-incubation. FIG. 15B shows a
similar reduction (p<0.05) after six hours co-incubation
followed by 18 hours incubation in fresh PYG medium.
[0279] The inhibitory effect of monoclonal antiidiotypic antibody
K20, tested over 6 days at 370.degree. C. in the same way as
described for KM, is shown in FIG. 16. The effect can be compared
to that of the rat monoclonal K10 (FIG. 17). After 6 days of
incubation, the number of trophozoites per well using K20 was lower
than when using K10 at 25 .mu.g (2700 vs. 3100) and 50 .mu.g (2800
vs. 3080).
[0280] Range of KM Activity
[0281] KM shows potent anti-C.albicans and anti-A.castellani
activity. KM was also found to be effective against other
microorganisms which are very important from an epidemiological
point of view, such as multidrug resistant strains of Candida spp.
and Mycobacterium tuberculosis, Cryptococcus neoformans (FIG. 10)
and Aspergillus fumigatus, but also against methicillin-resistant
strains of Staphylococcus aureus (FIG. 11), and
penicillin-resistant strains of Streptococcus pneumoniae.
[0282] Surprisingly, KM was also found to have anti-viral activity
against influenza A virus and HIV-1.
[0283] Influenza A Virus
[0284] The effect of KM on influenza virus replication was compared
to the scramble peptide SP control. As a further control,
replication in maintenance medium alone was tested.
[0285] Two different strains of type A influenza virus
(Ulster/73/H7N1, avian; NWS/33/H1N1, human neurovirulent) have been
previously demonstrated to efficiently replicate in LLC-MK2 (Rhesus
monkey kidney), MDCK (Madin Darby Canine Kidney) and AGMK-37RC
(African Green Monkey Kidney) cell cultures. Confluent monolayers
of the different cell lines were infected with the virus (moi=20
pfu, higher than a normal in vivo infection) in pre-warmed PBS (pH
7.4). After a 40 minute adsorption period, maintenance medium (MM)
containing either KM or SP was added to the cell cultures and virus
titre was determined after 24, 36, 48 or 72 hours of infection by
haemagglutinin (HA) titration in triplicate samples on cell
supernatant, after centrifugation at 2,000.times.g to remove
cellular debris.
[0286] The effect of KM and SP on HA titres 24 hours after
infection of LLC-MK2 cells by Ulster/73 is shown in FIG. 18. At
50.mu.g/ml or below, KM and SP had little or no effect on viral
growth. Above 60.mu.g/ml, however, KM peptide interferes with viral
growth, with complete blocking at 80 .mu.g/ml. Similar effects were
seen for the other virus, with the other cell lines, and at the
other time points. Thus KM can suppress viral replication in a
dose-dependent manner.
[0287] In a different set of experiments, fresh MM containing
either KM or SP was added to the cells at time 0 and then
substituted with the same peptide/medium mixture every 12 hours
starting at 24 hours and ending at 72 hours. KM at 80 .mu.g/ml was
able to completely block viral production at all time points
tested. Using SP, viral titres were similar to those obtained by
growth in MM only.
[0288] The effect of KM on influenza virus replication was also
tested using haemadsorption assays. HA is synthesised during
replication and inserted into the cell membrane before viral
budding. Haemadsorption of red blood cells to infected cells
correlates with the integrity of the glycosylated HA as well as
with its correct insertion into the cell membrane.
[0289] Cell cultures were infected, treated with 80 .mu.g/ml of
either KM or SP, as described above, and haemadsorption assays were
carried out after 48 hours. A significant reduction of OD.sub.420nm
was observed in infected cells treated with KM, demonstrating a
significant reduction of viral HA molecules on the plasma membrane
of those cells. Neither KM nor SP interferes with virus-specific
receptors on red blood cells (RBC).
[0290] Finally, the effect of laminarin (.beta.-1,3-glucan) on
anti-influenza activity was determined. As shown above, Laminarin
interferes with KM's candidacidal activity. KM or SP (80 .mu.g/ml)
was mixed with laminarin (320 .mu.g/ml) and the mixture was added
to infected cells at time 0, without preincubation or after 10
minutes of preincubation. Similar HA titres were obtained after 24
hours using KM/laminarin and SP/laminarin, suggesting that
laminarin abolishes the antiviral activity of KM. This effect was
not seen with pustulan.
[0291] HIV-1
[0292] The effect of KM on HIV-1 replication was compared to the
scramble peptide SP control. HIV-1 can replicate in peripheral
blood mononuclear cell (PBMC) cultures in the presence of exogenous
IL-2 [73]. To test KM activity, PBMC cultures were obtained from
patients in acute infection phase in whom HIV-1 proviral load was
known.
[0293] PBMCs were cultured in 48-well plates at the concentration
of 10.sup.6 cells/ml in RPMI 1640 medium supplemented with 10% FCS
in the presence of 50 U/ml of rIL-2. Half of the culture medium was
added at day 8. Supematants and cells were harvested for analysis
of HIV RNA and proviral contents, respectively. Exogenous rIL-2 was
added every 3 to 4 days, KM- or SP-peptide was added every 7 days.
HIV-1 expression was determined using HIV RNA branched kit (b-DNA,
Bayer.TM.), whereas HIV-1 proviral load was determined using
gene-detective HIV-1 gag EOA kit (ZeptoMetrix.TM.). For phenotype
determination, cells were analysed by flow cytometry after staining
with mAbs directed to CD antigens.
[0294] After PBMC isolation, the majority of PBMC of HIV cultures
were T-lymphocytes, as shown by the expression of CD3 Ag (CD4+=41%;
CD8+=36%). All primary PBMC cultures remained viable apparently for
at least 20 days. In the presence of exogenous IL-2, all
CD3+T-cells expressed CD25 and HLA-DR cell surface activation
markers after 7 days of culture.
[0295] Primary cultures were established from the patient's
cryo-preserved PBMC in the presence of 1.times. and 10.times.
concentration of KM peptide (1 .mu.g and 10 .mu.g, respectively).
As controls, SP peptide was used at the same concentrations. Other
control cultures (untreated cells) received medium only.
[0296] The kinetics of HIV RNA production at different time points
of PBMC cultures are shown in FIG. 19. The cultures showed early
peaks of viral copies within 5 to 10 days of culture which then
decreased in correspondence to the HIV-1-induced loss of CD4+
cells. HIV replication in these PBMC cultures was considerably
lower in the presence of KM (<44%, mean), with both
concentrations of KM having similar effects on the levels and
kinetics of HIV RNA production.
[0297] SP-treated cells showed the same levels and kinetics of HIV
RNA production as untreated controls.
[0298] D-Amino Acid Derivatives of KM and KM0
[0299] KM0 and KM were synthesised using the same amino acids
residues, but in the D- rather than L-conformation (KM6 and KM7).
Scramble peptide controls SP0 and SP were also synthesised using
D-amino acids.
[0300] KM6 and KM7 each showed candidacidal activity in the CFU
assay (FIG. 12). Activity was slightly lower than the L-amino acid
polypeptides, but this in vitro reduction does not take into
account the in vivo increase in half life which would be
expected.
[0301] A D-amino acid polypeptide with both the Glu.fwdarw.Ala and
Cys.fwdarw.Ser substitutions (SEQ ID 33) is useful.
[0302] Toxicity
[0303] Toxicity of KM was assessed by in vitro incubation with
LLC-MK2 rhesus monkey kidney cells. The cells were maintained in
Eagle's Minimum essential Medium (MEM) supplemented with 10% fetal
bovine serum, 100 U/ml penicillin and 100 .mu.g/ml streptomycin at
37.degree. C. in a humidified atmosphere containing 5% CO.sub.2.
The cells were plated in triplicate in 6-well dishes at
4.times.10.sup.4 cells per well and cultured for 24 hours. Serial
dilutions of peptides (final concentrations between 0 and 500
.mu.g/ml) in medium containing 10% fetal bovine serum were then
added to cells and incubated for 24 hours at 37.degree. C. Cells
were subsequently treated with MTT (50 .mu.g per well) and
incubated for another 2 hours. After solubilisation of the formazan
dye in DMSO, the absorbance of each well was measured at 550 and
620 nm. Peptide SP was also tested.
[0304] Cell viability as T/C % where T represents the mean
absorbance of the treated cells of the controls (FIG. 20).
[0305] The peptide displayed no toxic effects, even at 500
.mu.g/ml.
[0306] Equivalent Peptides From Within H20
[0307] An alignment of H6 and H20 sequences is given below, with
CDRs in bold: TABLE-US-00015 10 20 30 40 50 60 70 | | | | | | | H6
MAQVKLQESGPGLVAPSQSLSITCTVSGFSLTGYGVNWVRQPPGKGLEWLGMIWGD-GSTDYNSALKSRL
****:**:**. ** .. *:.::**.***.:..* ::**:* * :****:* * : *.*:* .
::.: H2O
MAQVQLQQSGAKLVRSGASVKLSCTTSGFNIKDYYMHWVKQRPEQGLEWIGWIDPENGDTEYAPKFQGKT
80 90 100 110 120 130 140 | | | | | | | H6
SISKDNSKSQVFLKMNSLQTDDTARYYC-------------LYAMDYWGQGTTVTCSSGGGGSGGGGSGG
::: *.*.. .:*::.** ::*** *** **************************** H2O
TLTADTSSNTAYLQLSSLTSEDTAVYYCNAWVYDGYSGDFYYYAMDYWGQGTTVTCSSGGGGSGGGGSGG
150 160 170 180 190 200 210 | | | | | | | H6
GGSDIELTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKPWIYLTSNLASGVPARFSGSGSG
************::*********:***********:*:**** :*** ***
****************** H2O
GGSDIELTQSPAIVSASPGEKVTITCSASSSVSYMHWFQQKPGTSPKLWIYSTSNLASGVPARFSGSGSG
220 230 240 250 260 | | | | | H6
TSYSLTISSMEAEDAATYYCQQWSSNPYTFGGGTKLEIKRAAAGAPVPYPDPLEPR ********
************* ** * *** ************************ H2O
TSYSLTISRMEAEDAATYYCQQRSSYPLTFGAGTKLEIKRAAAGAPVPYPDPLEPR
[0308] H20 peptide sequences corresponding to H6 peptides KM, KM0 ,
KM1, KM2, KM4 and KM5 are: TABLE-US-00016 H6 KM0 KM1 KM2 KM4 KM5 KM
H6 SEQ ID 3 23 24 26 27 4 H20 SEQ ID 34 35 36 37 38 39
[0309] It will be understood that the invention has been described
by way of example only and modifications may be made whilst
remaining within the scope and spirit of the invention.
REFERENCES (the contents of which are hereby incorporated in
full)
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Sequence CWU 1
1
71 1 756 DNA Artificial Sequence recombinant phage single chain Fv
H6 antibody nucleotide sequence 1 atggcccagg tgaagctgca ggagtctgga
cctggcctgg tggcgccctc acagagcctg 60 tccatcacat gcaccgtctc
agggttctca ttaaccggct atggtgtaaa ctgggttcgc 120 cagcctccag
gaaagggtct ggagtggctg ggaatgatat ggggtgatgg aagcacagac 180
tataattcag ctctcaaatc cagactgagc atcagcaagg acaactccaa gagccaagtt
240 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca ggtactactg
tctctatgct 300 atggactact ggggccaagg gaccacggtc accttctcct
caggtggagg cggttcaggc 360 ggaggtggct ctggcggtgg cggatcggac
atcgagctca ctcagtctcc agcactcatg 420 tctgcatctc caggggagaa
ggtcaccatg acctgcagtg ccagctcaag tgtaagttac 480 atgtactggt
accagcagaa gccaagatcc tcccccaaac cctggattta tctcacatcc 540
aacctggctt ctggagtccc tgctcgcttc agtggcagtg ggtctgggac ctcttactct
600 ctcacaatca gcagcatgga ggctgaagat gctgccactt attactgcca
gcagtggagt 660 agtaacccat acacgttcgg agggggcacc aagctggaaa
tcaaacgtgc ggccgcaggt 720 gcgccggtgc cgtatccgga tccgctggaa ccgcgt
756 2 252 PRT Artificial Sequence recombinant phage single chain Fv
H6 antibody amino acid sequence 2 Met Ala Gln Val Lys Leu Gln Glu
Ser Gly Pro Gly Leu Val Ala Pro 1 5 10 15 Ser Gln Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr 20 25 30 Gly Tyr Gly Val
Asn Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Leu
Gly Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala 50 55 60
Leu Lys Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val 65
70 75 80 Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg
Tyr Tyr 85 90 95 Cys Leu Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr
Thr Val Thr Cys 100 105 110 Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly 115 120 125 Ser Asp Ile Glu Leu Thr Gln Ser
Pro Ala Leu Met Ser Ala Ser Pro 130 135 140 Gly Glu Lys Val Thr Met
Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr 145 150 155 160 Met Tyr Trp
Tyr Gln Gln Lys Pro Arg Ser Ser Pro Lys Pro Trp Ile 165 170 175 Tyr
Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly 180 185
190 Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala
195 200 205 Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn
Pro Tyr 210 215 220 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Ala Ala Ala Gly 225 230 235 240 Ala Pro Val Pro Tyr Pro Asp Pro Leu
Glu Pro Arg 245 250 3 10 PRT Artificial Sequence synthetic H6 KM0,
residues 146-155 of scFv H6 (seq ID 2) 3 Glu Lys Val Thr Met Thr
Cys Ser Ala Ser 1 5 10 4 10 PRT Artificial Sequence synthetic H6
KM, Glu-Ala mutation of H6 KMO (seq ID 3) 4 Ala Lys Val Thr Met Thr
Cys Ser Ala Ser 1 5 10 5 10 PRT Artificial Sequence synthetic
alanine-scanning variant of H6 KM0 (seq ID 3) 5 Glu Ala Val Thr Met
Thr Cys Ser Ala Ser 1 5 10 6 10 PRT Artificial Sequence synthetic
alanine-scanning variant of H6 KM0 (seq ID 3) 6 Glu Lys Ala Thr Met
Thr Cys Ser Ala Ser 1 5 10 7 10 PRT Artificial Sequence synthetic
alanine-scanning variant of H6 KM0 (seq ID 3) 7 Glu Lys Val Ala Met
Thr Cys Ser Ala Ser 1 5 10 8 10 PRT Artificial Sequence synthetic
alanine-scanning variant of H6 KM0 (seq ID 3) 8 Glu Lys Val Thr Ala
Thr Cys Ser Ala Ser 1 5 10 9 10 PRT Artificial Sequence synthetic
alanine-scanning variant of H6 KM0 (seq ID 3) 9 Glu Lys Val Thr Met
Ala Cys Ser Ala Ser 1 5 10 10 10 PRT Artificial Sequence synthetic
alanine-scanning variant of H6 KM0 (seq ID 3) 10 Glu Lys Val Thr
Met Thr Ala Ser Ala Ser 1 5 10 11 10 PRT Artificial Sequence
synthetic alanine-scanning variant of H6 KM0 (seq ID 3) 11 Glu Lys
Val Thr Met Thr Cys Ala Ala Ser 1 5 10 12 10 PRT Artificial
Sequence synthetic alanine-scanning variant of H6 KM0 (seq ID 3) 12
Glu Lys Val Thr Met Thr Cys Ser Ala Ala 1 5 10 13 10 PRT Artificial
Sequence synthetic SP0 scramble control peptide 13 Met Ser Thr Ala
Val Ser Lys Cys Glu Thr 1 5 10 14 9 PRT Artificial Sequence
synthetic shortened KM-derived peptide (9 mers of seq ID 4) 14 Ala
Lys Val Thr Met Thr Cys Ser Ala 1 5 15 8 PRT Artificial Sequence
synthetic shortened KM-derived peptide (8 mers of seq ID 4) 15 Ala
Lys Val Thr Met Thr Cys Ser 1 5 16 7 PRT Artificial Sequence
synthetic shortened KM-derived peptide (7 mers of seq ID 4) 16 Ala
Lys Val Thr Met Thr Cys 1 5 17 6 PRT Artificial Sequence synthetic
shortened KM-derived peptide (6 mers of seq ID 4) 17 Ala Lys Val
Thr Met Thr 1 5 18 5 PRT Artificial Sequence synthetic shortened
KM-derived peptide (5 mers of seq ID 4) 18 Ala Lys Val Thr Met 1 5
19 4 PRT Artificial Sequence synthetic shortened KM-derived peptide
(4 mers of seq ID 4) 19 Ala Lys Val Thr 1 20 3 PRT Artificial
Sequence synthetic shortened KM-derived peptide (3 mers of seq ID
4) 20 Ala Lys Val 1 21 798 DNA Artificial Sequence recombinant
phage single chain Fv H20 antibody nucleotide sequence 21
atggcccagg tgcagctgca gcagtctggg gcaaagcttg tgaggtcagg ggcctcagtc
60 aagttgtcct gcacaacttc tggcttcaac attaaagact actatatgca
ctgggtgaag 120 caaaggcctg aacagggcct ggagtggatt ggatggattg
atcctgagaa tggtgatact 180 gaatatgccc cgaagttcca gggcaagacc
actctgactg cagacacatc ctccaacaca 240 gcctacctgc agctcagcag
cctgacatct gaggacactg ccgtctatta ctgtaatgca 300 tgggtctatg
atggttactc gggtgatttt tattactatg ctatggacta ctggggccaa 360
gggaccacgg tcaccttctc ctcaggtgga ggcggttcag gcggaggtgg ctctggcggt
420 ggcggatcgg acatcgagct cactcagtct ccagcaatcg tgtctgcatc
tccaggggag 480 aaggtcacca taacctgcag tgccagctca agtgtaagtt
acatgcactg gttccagcag 540 aagccaggca cttctcccaa actctggatt
tatagcacat ccaacctggc ttctggagtc 600 cctgctcgct tcagtggcag
tggatctggg acctcttact ctctcacaat cagccgaatg 660 gaggctgaag
atgctgccac ttattactgc cagcaaagga gtagttaccc gctcacgttc 720
ggtgctggca ccaagctgga aatcaaacgt gcggccgcag gtgcgccggt gccgtatccg
780 gatccgctgg aaccgcgt 798 22 266 PRT Artificial Sequence
recombinant phage single chain Fv H20 antibody amino acid sequence
22 Met Ala Gln Val Gln Leu Gln Gln Ser Gly Ala Lys Leu Val Arg Ser
1 5 10 15 Gly Ala Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn
Ile Lys 20 25 30 Asp Tyr Tyr Met His Trp Val Lys Gln Arg Pro Glu
Gln Gly Leu Glu 35 40 45 Trp Ile Gly Trp Ile Asp Pro Glu Asn Gly
Asp Thr Glu Tyr Ala Pro 50 55 60 Lys Phe Gln Gly Lys Thr Thr Leu
Thr Ala Asp Thr Ser Ser Asn Thr 65 70 75 80 Ala Tyr Leu Gln Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Asn Ala
Trp Val Tyr Asp Gly Tyr Ser Gly Asp Phe Tyr Tyr 100 105 110 Tyr Ala
Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Cys Ser Ser 115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 130
135 140 Ile Glu Leu Thr Gln Ser Pro Ala Ile Val Ser Ala Ser Pro Gly
Glu 145 150 155 160 Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val
Ser Tyr Met His 165 170 175 Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro
Lys Leu Trp Ile Tyr Ser 180 185 190 Thr Ser Asn Leu Ala Ser Gly Val
Pro Ala Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Ser Tyr Ser
Leu Thr Ile Ser Arg Met Glu Ala Glu Asp 210 215 220 Ala Ala Thr Tyr
Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Leu Thr Phe 225 230 235 240 Gly
Ala Gly Thr Lys Leu Glu Ile Lys Arg Ala Ala Ala Gly Ala Pro 245 250
255 Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 260 265 23 10 PRT
Artificial Sequence synthetic H6 KM1, Cys-Ser mutation of H6 KMO
(Seq ID 3) 23 Glu Lys Val Thr Met Thr Ser Ser Ala Ser 1 5 10 24 10
PRT Artificial Sequence synthetic H6 KM2, residues 91-100 of scFv
H6 (Seq ID 2) 24 Asp Thr Ala Arg Tyr Tyr Cys Leu Tyr Ala 1 5 10 25
28 PRT Artificial Sequence synthetic H6 KM3 (H6 KMO + linker + H6
KM2) 25 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Gly Gly Gly Gly
Gly 1 5 10 15 Gly Ser Asp Thr Ala Arg Tyr Tyr Cys Leu Tyr Ala 20 25
26 15 PRT Artificial Sequence synthetic H6 KM4, residues 146-160 of
scFv H6 (Seq ID 2) 26 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr 1 5 10 15 27 9 PRT Artificial Sequence synthetic H6
KM5, residues 155-163 of scFv H6 (Seq ID 2) 27 Ser Ser Ser Val Ser
Tyr Met Tyr Trp 1 5 28 9 PRT Artificial Sequence synthetic
irrelevant control peptide (IP) 28 Thr Ser Thr Thr Ser Leu Glu Leu
Asp 1 5 29 10 PRT Artificial Sequence synthetic SP scramble control
peptide 29 Met Ser Thr Ala Val Ser Lys Cys Ala Thr 1 5 10 30 8 PRT
Artificial Sequence synthetic Linker used to join H6 KMO and H6 KM2
to give H6 KM3 30 Ser Gly Gly Gly Gly Gly Gly Ser 1 5 31 9 PRT
Artificial Sequence synthetic Peptide control 31 Tyr Met Trp Tyr
Thr Trp Gly Thr Gly 1 5 32 10 PRT Artificial Sequence synthetic H6
KM derivative (Cys-Xaa mutation of Seq ID 4) 32 Ala Lys Val Thr Met
Thr Xaa Ser Ala Ser 1 5 10 33 10 PRT Artificial Sequence synthetic
H6 KM derivative (Seq ID 32 with Xaa = Ser) 33 Ala Lys Val Thr Met
Thr Ser Ser Ala Ser 1 5 10 34 10 PRT Artificial Sequence synthetic
H20 KM0, residues 160-169 of H20 (Seq ID 22) 34 Glu Lys Val Thr Ile
Thr Cys Ser Ala Ser 1 5 10 35 10 PRT Artificial Sequence synthetic
H20 KM1, Cys-Ser mutation of H20 KMO (Seq ID 34) 35 Glu Lys Val Thr
Ile Thr Ser Ser Ala Ser 1 5 10 36 10 PRT Artificial Sequence
synthetic H20 KM2, residues 91-100 of H20 (Seq ID 22) 36 Asp Thr
Ala Val Tyr Tyr Cys Asn Ala Trp 1 5 10 37 15 PRT Artificial
Sequence synthetic H20 KM4, residues 160-174 of H20 (Seq ID 22) 37
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr 1 5 10
15 38 9 PRT Artificial Sequence synthetic H20 KM5, residues 169-177
of H20 (Seq ID 22) 38 Ser Ser Ser Val Ser Tyr Met His Trp 1 5 39 10
PRT Artificial Sequence synthetic H20 KM, Glu-Ala mutation of H 20
KMO (Seq ID 34) 39 Ala Lys Val Thr Ile Thr Cys Ser Ala Ser 1 5 10
40 10 PRT Artificial Sequence synthetic H6 KM2-derived peptide,
alanine-scanning variant of H6 KM2 (seq ID 24) 40 Ala Thr Ala Arg
Tyr Tyr Cys Leu Tyr Ala 1 5 10 41 10 PRT Artificial Sequence
synthetic H6 KM2-derived peptide, alanine-scanning variant of H6
KM2 (seq ID 24) 41 Asp Ala Ala Arg Tyr Tyr Cys Leu Tyr Ala 1 5 10
42 10 PRT Artificial Sequence synthetic H6 KM2-derived peptide,
alanine-scanning variant of H6 KM2 (seq ID 24) 42 Asp Thr Ala Ala
Tyr Tyr Cys Leu Tyr Ala 1 5 10 43 10 PRT Artificial Sequence
synthetic H6 KM2-derived peptide, alanine-scanning variant of H6
KM2 (seq ID 24) 43 Asp Thr Ala Arg Ala Tyr Cys Leu Tyr Ala 1 5 10
44 10 PRT Artificial Sequence synthetic H6 KM2-derived peptide,
alanine-scanning variant of H6 KM2 (seq ID 24) 44 Asp Thr Ala Arg
Tyr Ala Cys Leu Tyr Ala 1 5 10 45 10 PRT Artificial Sequence
synthetic H6 KM2-derived peptide, alanine-scanning variant of H6
KM2 (seq ID 24) 45 Asp Thr Ala Arg Tyr Tyr Ala Leu Tyr Ala 1 5 10
46 10 PRT Artificial Sequence synthetic H6 KM2-derived peptide,
alanine-scanning variant of H6 KM2 (seq ID 24) 46 Asp Thr Ala Arg
Tyr Tyr Cys Ala Tyr Ala 1 5 10 47 10 PRT Artificial Sequence
synthetic H6 KM2-derived peptide, alanine-scanning variant of H6
KM2 (seq ID 24) 47 Asp Thr Ala Arg Tyr Tyr Cys Leu Ala Ala 1 5 10
48 10 PRT Artificial Sequence synthetic H6 KM2-derived peptide,
Cys-Ser mutation of H6 KM2 (seq ID 24) 48 Asp Thr Ala Arg Tyr Tyr
Ser Leu Tyr Ala 1 5 10 49 9 PRT Artificial Sequence synthetic H6
KM5-derived peptide, alanine-scanning variant of H6 KM5 (seq ID 27)
49 Ala Ser Ser Val Ser Tyr Met Tyr Trp 1 5 50 9 PRT Artificial
Sequence synthetic H6 KM5-derived peptide, alanine-scanning variant
of H6 KM5 (seq ID 27) 50 Ser Ala Ser Val Ser Tyr Met Tyr Trp 1 5 51
9 PRT Artificial Sequence synthetic H6 KM5-derived peptide,
alanine-scanning variant of H6 KM5 (seq ID 27) 51 Ser Ser Ala Val
Ser Tyr Met Tyr Trp 1 5 52 9 PRT Artificial Sequence synthetic H6
KM5-derived peptide, alanine-scanning variant of H6 KM5 (seq ID 27)
52 Ser Ser Ser Ala Ser Tyr Met Tyr Trp 1 5 53 9 PRT Artificial
Sequence synthetic H6 KM5-derived peptide, alanine-scanning variant
of H6 KM5 (seq ID 27) 53 Ser Ser Ser Val Ala Tyr Met Tyr Trp 1 5 54
9 PRT Artificial Sequence synthetic H6 KM5-derived peptide,
alanine-scanning variant of H6 KM5 (seq ID 27) 54 Ser Ser Ser Val
Ser Ala Met Tyr Trp 1 5 55 9 PRT Artificial Sequence synthetic H6
KM5-derived peptide, alanine-scanning variant of H6 KM5 (seq ID 27)
55 Ser Ser Ser Val Ser Tyr Ala Tyr Trp 1 5 56 9 PRT Artificial
Sequence synthetic H6 KM5-derived peptide, alanine-scanning variant
of H6 KM5 (seq ID 27) 56 Ser Ser Ser Val Ser Tyr Met Ala Trp 1 5 57
9 PRT Artificial Sequence synthetic H6 KM5-derived peptide,
alanine-scanning variant of H6 KM5 (seq ID 27) 57 Ser Ser Ser Val
Ser Tyr Met Tyr Ala 1 5 58 15 PRT Artificial Sequence synthetic H6
KM4-derived peptide, Cys-Ser mutation of H6 KM4 (seq ID 26) 58 Glu
Lys Val Thr Met Thr Ser Ser Ala Ser Ser Ser Val Ser Tyr 1 5 10 15
59 13 PRT Artificial Sequence synthetic C-terminus E-tag system for
scFv 59 Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg 1 5 10
60 5 PRT Artificial sequence synthetic H6 CDR-H1, residues 33-37 of
H6 (Seq ID 2) 60 Gly Tyr Gly Val Asn 1 5 61 14 PRT Artificial
sequence synthetic H6 CDR-H2, residues 52-65 of H6 (Seq ID 2) 61
Met Ile Trp Gly Asp Gly Ser Thr Asp Tyr Asn Ser Ala Leu 1 5 10 62 4
PRT Artificial sequence synthetic H6 CDR-H3, residues 98-101 of H6
(Seq ID 2) 62 Leu Tyr Ala Met 1 63 10 PRT Artificial sequence
synthetic H6 CDR-L1, residues 153-162 of H6 (Seq ID 2) 63 Ser Ala
Ser Ser Ser Val Ser Tyr Met Tyr 1 5 10 64 7 PRT Artificial sequence
synthetic H6 CDR-L2, residues 178-184 of H6 (Seq ID 2) 64 Leu Thr
Ser Asn Leu Ala Ser 1 5 65 8 PRT Artificial sequence synthetic H6
CDR-L3, residues 217-224 of H6 (Seq ID 2) 65 Gln Gln Trp Ser Ser
Asn Pro Tyr 1 5 66 5 PRT Artificial sequence synthetic H20 CDR-H1,
residues 33-37 of H20 (Seq ID 22) 66 Asp Tyr Tyr Met His 1 5 67 15
PRT Artificial sequence synthetic H20 CDR-H2, residues 52-66 of H20
(Seq ID 22) 67 Trp Ile Asp Pro Glu Asn Gly Asp Thr Glu Tyr Ala Pro
Lys Phe 1 5 10 15 68 17 PRT Artificial sequence synthetic H20
CDR-H3, residues 99-115 of H20 (Seq ID 22) 68 Asn Ala Trp Val Tyr
Asp Gly Tyr Ser Gly Asp Phe Tyr Tyr Tyr Ala 1 5 10 15 Met 69 10 PRT
Artificial sequence synthetic H20 CDR-L1, residues 167-176 of H20
(Seq ID 22) 69 Ser Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 70 7
PRT Artificial sequence synthetic H20 CDR-L2, residues 192-198 of
H20 (Seq ID 22) 70 Ser Thr Ser Asn Leu Ala Ser 1 5 71 8 PRT
Artificial sequence synthetic H20 CDR-L3, residues 231-238 of H20
(Seq ID 22) 71 Gln Gln Arg Ser Ser Tyr Pro Leu 1 5
* * * * *
References